Microscopy, Cell, and Membrane PDF

Microscopy All organisms are composed of one or more cells. Cells are the smallest living units of all living organisms. Cells arise only by division of a previously existing cell.  Microscopy Magnification - how much bigger a sample appears to b...

Microscopy All organisms are composed of one or more cells. Cells are the smallest living units of all living organisms. Cells arise only by division of a previously existing cell.  Microscopy Magnification - how much bigger a sample appears to be under the microscope than it is in real life. Overall magnification = Objective lens x Eyepiece lens Resolution - the ability to distinguish between two points on an image i.e. the amount of detail. Resolution: (a) The two dots are resolved – that is, they can clearly be seen as separate structures. (b) These two dots are not resolved – they appear to be fused.  Microscopy Common types of microscopes: 1. Light (optical) microscopes 2. Electron microscopes The world's first practical microscope: Antony Van Leeuwenhoek (1632- 1723) Light Microscope Simplest and most widely-used Specimens are illuminated with light, which is focused using glass lenses and viewed using the eye or photographic film. Specimens can be living or dead, but often need to be stained with a coloured dye to make them visible. Compound Light Microscope All light microscopes are compound microscopes - uses several lenses to obtain high magnification. Eyepiece/Optical lens – 10x Objective lens – 4 x 10 x 40 x 100 x Total Magnification - how much bigger a sample appears to be under the microscope than it is in real life. Overall magnification = Objective lens x Eyepiece lens Light microscopy has a resolution of about 200 nm, which is good enough to see cells, but not the details of cell organelles.  Measuring Cells Cells and organelles structure can be measured accurately - suitable scale: Eye-piece graticule – Transparent scale, generally with 100 divisions placed in the microscope eyepiece Stage micrometer - Miniature transparent ruler placed on the microscope stage Magnification = Size of image Actual size of the specimen Measuring Cells  Measuring Cells The images of the two scales can be then superimposed as shown below: a b c 100 eyepiece graticule divisions = 0.25 mm Hence, 1 eyepiece graticule division = 0.25/100 = 0.0025 mm or 2.5 µm Diameter of the cell shown = 20 * 2.5µm = 50 µm Measuring Cells Measuring Cells  Electron Microscopy Principles Electron Microscope Uses a beam of electrons to "illuminate" the specimen. A beam of electrons has an effective wavelength of less than 1 nm, so can be used to resolve small sub-cellular ultrastructure. Limitations: (i) specimens must be fixed in plastic and viewed in a vacuum, and must therefore be dead; (ii) specimens can be damaged by the electron beam; (iii) must be stained with an electron-dense chemical (usually heavy metals like osmium, lead or gold). Two Types: - Transmission electron microscope (TEM) - Scanning electron microscope (SEM) 1. Transmission electron microscope (TEM) Transmits a beam of electrons through a thin specimen and then focusing the electrons to form an image on a screen or on film. Most common form of electron microscope and has the best resolution. 2. Scanning electron microscope (SEM) Scans a fine beam of electron onto a specimen and collects the electrons scattered by the surface. Has poorer resolution, but gives excellent 3-dimentional images of surfaces.  Light Microscope Electron Microscope Cheap to purchase (RM400 – 2000) Expensive to buy (over RM5000 000) Cheap to operate Expensive to produce electron beam Small and portable Large and requires special rooms Simple and easy sample preparation Lengthy and complex sample prep. Material rarely distorted by preparation Preparation distorts material Vacuum is not required Vacuum is required Natural colour of sample maintained All images in black and white Magnifies objects only up to 2000 Magnifies over 500 000 times times Specimens are dead, as they must be Specimens can be living or dead fixed in plastic and viewed in a vacuum The electron beam can damage specimens and they must be Stains are often needed to make the stained with an electron-dense cells visible chemical (usually heavy metals like osmium, lead or gold)  Label the components of the light microscope. 1 7 2 1 3 8 4 9 5 10 6 2  Prokaryote and Eukaryote The Cell Theory The initial development of the theory, during the mid-17th century, was made possible by advances in microscopy The three parts to the cell theory are as described below: 1. All living organisms are composed of one or more cells. 2. The cell is the basic unit of structure, function, and organization in all organisms. 3. All cells come from preexisting, living cells.  Development of Cell Theory 1838 1839 Matthias Schleiden –Cell theory; Plants are made of cells. Theodor Schwann – Cell theory; Animals are made of cells. 1855 Rudolf Virchow (1855) – New cells come from existing cells. MAZARELLO, P. (1999). A unifying concept: the history of cell theory Nature Cell Biology, 1: E13-E15 What is a cell? Cells are the basic unit of living things. All living organisms are made of cells. A cell is a small, membrane enclosed structure filled with an aqueous solution where organelles and other subcellular structures are found. Two types of cells make up every organism: - prokaryotic cells - eukaryotic cells Is virus a cell? Features Common To All Cells All membranes have the same lipid bilayer. All cells use membranes to form boundaries. Membranes are semi-permeable barriers controls the passage of molecules. Membranes The membranes separate the cell contents from the external environment. All cells store genetic information as DNA. Genetic material Central dogma (information flows from DNA to protein); Replication, Transcription, Translation. Cytoplasm Cytoplasm is the jelly-like material filling the cell interior  Do not contain a nucleus. Have their DNA located in a region called the nucleoid. No membrane bound Prokaryotic cells organelles. Contain a true nucleus, bounded by a membranous nuclear envelope. Generally quite a bit bigger than prokaryotic cells. Contain membrane bound organelles. Eukaryotic cells  Pili: attachment structures on the surface of some prokaryotes Nucleoid: region where the cell’s DNA is located (not enclosed by a membrane) Ribosomes: organelles that synthesize proteins Plasma membrane: membrane enclosing the cytoplasm Cell wall: rigid structure outside the plasma membrane Capsule: jelly-like outer coating Bacterial of many prokaryotes chromosome 0.5 µm (a) A typical Flagella: locomotion (b) A thin section through the rod-shaped bacterium organelles of bacterium Bacillus coagulans some bacteria (TEM) * Plasma membrane of prokaryotic cell fold inwards to form a structure called mesosome. Mesosome could function Prokaryotic cell as respiratory site / photosynthetic site.  In animal cells but not plant cells: Lysosomes Eukaryotic cell: an animal cell Centrioles Flagella (in some plant sperm)  In plant cells but not animal cells: Chloroplasts Eukaryotic cell: a plant cell Central vacuole and tonoplast Cell wall Plasmodesmata Characteristics Prokaryotic cell Eukaryotic cell Type of organism Bacteria & cyanobacteria Algae, fungi, protoctist, animals & plants Size range Small ( 0.5 – 10.0 μm ) in diameter Larger, ( 10 – 100 μm in diameter) Nucleus No distinct nucleus, diffused area of A distinct membrane bound nucleus nucleoplasm with no nuclear envelope Chromosomes None, has cicular strands of DNA in the Present nucleiod region Ribosomes Protein synthesized by smaller ( 70s) Protein synthesized by larger (80s) ribosomes that occur as free particles in ribosomes that occur as free particles in the cytoplasm the cytoplasm or bound endoplasmic reticulum Membrane None Present bound organelle Chloroplast None, only photosynthetic lamellae Present in algae and plant cells present in cyanobacteria Nuclear division No mitosis/meiosis, only binary fission, Mitosis and meiosis occur, there is spindle no spindle formation formation Cell wall Consists of peptidoglycan Plant cell – cellulose Fungal cell – chitin Animal cells do not have cell walls Plasmids Present in some cells Absent  The Organelles Nucleus Double Mitochondria membranes Chloroplast Membrane Endoplasmic reticulum Single membrane Golgi apparatus Organelles Lysosomes Vacuole Ribosomes No membrane Centrioles Flagellum & cilium Cytoskeleton  Nucleus Found in all cells except prokaryotic cells, red blood cells and mature sieve tubes. The largest organelle and roughly spherical in shape. The nucleus is the control centre of the cell and the site where hereditary information is stored. Inside the nucleus there is a dark region called nucleolus, where you can find chromatins. The nucleolus is not bounded by a membrane.  Nucleus Nucleoplasm 1 µm Nucleus Nucleolus Chromatin Nuclear envelope: Inner membrane Outer membrane Nuclear pore Pore complex Rough ER Surface of nuclear envelope Ribosome 1 µm 0.25 µm Close-up of nuclear envelope Pore complexes (TEM) Nuclear lamina (TEM) The nucleolus is comprised of granular and fibrillar components, as well as an ill-defined matrix, in addition to DNA. The granular material consists of ribosomal subunits that have already been formed but have not yet matured and are waiting to be exported to the cytoplasm.  The nucleolus is also where rRNA and ribosome are made. The surface of the nucleus is bounded by double membrane, make up the nuclear envelope. The outer membrane of the nuclear envelope is continuous with a membrane system called endoplasmic reticulum.  On the surface of the nuclear envelope are nuclear pores (40- 100nm diameter, 3000/nucleus).These are found where the two membranes pinch together. Nuclear pores are lined with proteins. They act as molecular channels and only two types of molecules are given passage: - proteins associated with nuclear structures or nuclear activities. - RNA and protein-RNA complexes  The space between the nuclear envelope and nucleolus is called the nucleoplasm. It is a semi-fluid medium and it contains chromatins. Chromatins are chromosomes that are in their non-dividing state. They have a thread-like appearance. Chromatin contains genetic code that controls cell and it made of DNA & proteins. Simon et al. (2009). Cambell Essential Biology, Pearson; 4th edition  During cell division, chromatins condense to form thick rod-like structures called chromosomes. Chromatin is made up of DNA and protein (histones) and has no particular shape. In a non-dividing cell, the nucleus has a ‘grainy’ appearance. This is due to the presence of chromatins. Function of Nucleus To contain the genetic material of a cell in the form of chromosomes. As a control centre for the activities of a cell. To carry the instructions for the synthesis of protein in the nuclear DNA. Involved in the production of ribosomes and RNA.  Mitochondria Distributed in all cells except red blood cells. Distributed randomly in cytoplasm. Shape: vary, might be oval, rod, sphere. ~ 2.5µm in length and ~ 1µm in diameter. Function: generate ATP during aerobic respiration. Bound by two membranes: outer membrane and inner membrane. The outer membrane is smooth.  The inner membrane is folded to form a projection structures called cristae. The surface of the inner membrane is embedded with proteins that carry out oxidative metabolism. Many stalked particles which contain ATPase enzyme are found in inner membrane Matrix mitochondria contains DNA, ribosome, RNA, respiration enzyme, proteins and etc.  Mitochondrion Intermembrane space Outer membrane Free ribosomes in the mitochondrial matrix Inner membrane Cristae Matrix Mitochondrial 100 µm DNA  The space between the inner membrane and the outer membrane is called the intermembrane space. Mitochondria has its own DNA. It is not replicated through cell division but replicates by itself. Meaning, a parent mitochondria divides into two daughter mitochondria to multiply. However, mitochondria is not able to replicate independently, outside the cell. Chloroplast Distributed at high level plants. Can be found at mesophyll especially palisade mesophyll. Plastids are organelles that conduct photosynthesis and store starch. Diameter: 8-10µm. Stroma is the matrix of chloroplast. It contains starch, DNA, lipid globule, ribosome and photosynthetic enzyme.  Chloroplast Ribosomes Stroma Chloroplast Inner and outer DNA membranes Granum 1 µm Thylakoid  Chloroplasts have outer membrane and inner membrane. A closed compartment of stacked membranes called grana (granum). The granum may be made up of many stacked, disk- shaped structures called thylakoids. A granum comprises of 2 – 100 thylakoids. Chloroplasts contain the photosynthetic pigment called chlorophyll which located in the thylakoid membranes. Functions of Chloroplast Carry out photosynthesis. Storage of photosynthesis products. Chloroplast contain DNA that involved in genetic transfer.  Vacuole A sac surrounded by a single lipoprotein membrane; spherical in shape. The lipoprotein membrane surrounding the vacuole is called tonoplast. It contains a solution of water, sugars, ions and pigments called the cell sap. There are 3 types of vacuole: i. sap or central vacuole; ii. food vacuole; iii. contractile vacuole. Functions of Vacuole Storage centre for the important substances. Gives shape and support to the cell. Maintains water and salt balance of the cell. Increases the surface-to-volume ratio of the plant cell. Stores cell’s waste products Contributes to the growth of the cell by taking in water and enlarging.  Endoplasmic Reticulum It is an extension of the outer nuclear membrane with which it is continuous. Membranes forms a series of sheets which enclose flattened sacs called cisternae. It is the site of the synthesis of many substances in the cell. ER can be found in large numbers at secretory cells, liver cells, pancreatic cells, brain cells, and testiscular cells.  Smooth ER Rough ER Nuclear envelope ER lumen Cisternae Ribosomes Transport vesicle 200 µm Smooth ER Rough ER  The rough ER is studded with 80S ribosomes and is the site of protein synthesis. It is an extension of the outer membrane of the nuclear envelope, so allowing mRNA to be transported swiftly into 80S ribosomes, where they are translated in protein synthesis. Smooth ER is a network of tubules without ribosome on its surface.  Smooth ER is the site of lipid and steroid synthesis, and is associated with the Golgi apparatus. Smooth ER also involved in the regulation of calcium levels in muscle cells, and the breakdown of toxins by liver cells. Damage to a cell often results in increased formation of ER in order to produce the protein necessary for cell repair. Smooth endoplasmic Rough endoplasmic reticulum reticulum Functions of ER ▪ Smooth ER – synthesis of lipid, steroids, cholesterols and sex hormones. ▪ Smooth ER – help detoxify drugs and poisons, especially in liver cells. ▪ Rough ER – synthesis of protein. ▪ Works as intracellular transportation system – lipids, cholesterol, proteins are transported through ER. ▪ Provides a large surface area for biochemical reactions in cells. ▪ Most enzyme are inactivate in cytoplasm. ER provides surface for enzymes during chemical reactions. ▪ As a part of the cytoskeleton structure to maintain the shape of the cell.  Ribosomes Distribution: rough ER, cytoplasm of prokaryote, mitochondria, chloroplast. Ribosomes are tiny granule-like organelles that conduct protein synthesis. There are 2 major types: 80S (found in eukaryotes) and 70S (found in prokaryotes). They are found abundantly in cells that produce a lot of proteins such as pancreatic cells and liver cells. Ribosomes Cytosol Free ribosomes Bound ribosomes Large subunit Small 0.5 µm subunit TEM showing ER and ribosomes Diagram of a ribosome  A typical bacterial cell contains about 10, 000 ribosomes. Each ribosome is made up of a small subunit and a large subunit. 80S → 60S + 40S 70S → 50S + 30S The small and large subunits combine in the presence of magnesium ions to produce one functional unit.  Both subunits are made from rRNA and proteins. They are both synthesized in the nucleolus. Chloroplasts and mitochondria have the 70S type of ribosomes. This proves that chloroplast and mitochondria originated from prokaryotes. Ribosomes are the site for protein synthesis.  Proteins that are synthesized on the free ribosomes are NOT destined for export. They are released in the cytosol and are for the use of the cell. Proteins synthesized by the ribosomes on the ER are passed into the ER cisternae and transported to the Golgi apparatus. These proteins are then secreted from the cell as digestive enzymes or hormones. Functions of Ribosomes Synthesis protein Synthesis enzyme  Golgi Apparatus Distributed widely at series of gland cells e.g. nerve cells, pancreatic cells. Composed of stacks of flattened sacs made of membranes (~ 5-6 cisternae in a Golgi apparatus). The sacs are fluid filled and pinch off smaller membranous sacs, called vesicles, at their ends. All proteins produced by the endoplasmic reticulum are passed through the Golgi apparatus in a strict sequence.  The GA has 2 faces: cis face (located near the ER and nucleus) and trans face (located further away from the nucleus). They pass first through the cis- golgi network, which returns to the ER if any proteins wrongly exported by it. They then pass through the stack of cisternae, which modify the proteins and lipids undergoing transport and add labels which allow them to be identified and sorted at the next stage, the trans-Golgi network.  Here the proteins and lipids are sorted and sent to the final destinations. Movement between cisternae is by means of vesicles that bud off from one cisternae and fuse with the nest. The final products then packaged into secretory vesicles. This vesicle buds off at the trans face.  The secretory vesicles will be delivered to: - other organelles such as lysosomes - the plasma membrane, so that the contents of the vesicle can be released to the outside of the cell through exocytosis. Golgi acts as the cell’s post office, receiving, sorting and delivering proteins, lipids, carbohydrates, enzymes, steroids and etc.  cis face – (receiving side of Golgi apparatus) 1 Vesicles move 2 Vesicles coalesce to 0.1 0 µm 6 Vesicles also from ER to Golgi form new cis Golgi cisternae transport certain proteins back to ER Cisternae 3 Cisternal maturation: Golgi cisternae move in a cis- to-trans direction 4 Vesicles form and leave Golgi, carrying specific proteins to other locations or to the plasma mem- brane for secretion 5 Vesicles also transport trans face – specific proteins backward (“shipping” side of TEM of Golgi apparatus to newer Golgi cisternae Golgi apparatus) Functions of Golgi Apparatus Stores and processes proteins, lipids and carbohydrates – form glycoprotein, lipoprotein and glycolipid. Important intracellular transportation system – collect, pack and transport the synthesis substances from within the cells. Excrete the synthesis substances in the cells through secretory vesicles.  Forming lysosome – the hydrolytic enzyme is synthesised by ribosomes and transported to Golgi apparatus to be packed in vesicles that is known as lysosomes. Maintain and increase the plasma membrane – excretion vesicles from Golgi apparatus are a part of plasma membrane. Relationships among organelles of the endomembrane system  Lysosome Spherical bodies, diameter: 0.1-1.0µm Found in animal cells; not found in plant cell except certain species such as Nephentes. White blood cells especially macrophage contains a lot of lysosomes. Lysosomes contain around 50 enzymes, their contents are generally acidic. Main components of lysosome is hydrolytic enzymes and proteins.  The single membrane of lysosome can resist against the digestive enzyme such as hydrolytic enzyme in lysosome and the membrane is not permeable to lysosomal enzyme. They isolate the hydrolytic enzymes from the remainder of the cell and by doing so prevent them from acting upon other chemicals and organelles within the cells. Functions of Lysosomes Digest material which the cell consumes from the environment. Autophagy – digest parts of the cell, such as worn-out organelles. Autolysis – after the death of the cell, the lysosome are responsible for its complete breakdown. Release their enzymes outside the cell in order to break down other cells  2 3 4 1 Functions of lysosomes: 1. Digestion of ingested material 2. Autophagy 3. Cell death 4. Extracellular function Phagocytosis is the ingestion of solid particles by endocytosis. The cytoplasmic membrane invaginates and pinches off placing the particle in a phagocytic vacuole. The phagocytic vacuole then fuses with lysosomes and the material is degraded.  1 µm Lysosome containing Nucleus two damaged organelles 1µm Mitochondrion fragment Peroxisome Lysosome fragment Lysosome contains Food vacuole Hydrolytic Lysosome fuses with Hydrolytic enzymes active hydrolytic fuses with enzymes digest vesicle containing digest organelle enzymes lysosome food particles damaged organelle components Digestive enzymes Lysosome Lysosome Plasma membrane Digestion Food vacuole Digestion Vesicle containing damaged mitochondrion (a) Phagocytosis: lysosome digesting food (b) Autophagy: lysosome breaking down damaged organelle  Centrosome Found only in animal cells. A region of cell where microtubules radiate. Centrosome is a clear part of cytoplasm near the nucleus, inside this centrosome is a pair of centrioles. Centrioles are composed of microtubules in triplet arrangement. Centrioles make up of 9 sets of triplet microtubules and has the “9+0” pattern. It organizes microtubules that attach to chromosomes during cell division.  Centrosome Microtubule Centrioles 0.25 µm Centriole: “9 + 0” pattern ring Longitudinal section Microtubules Cross section of one centriole of the other centriole Cilia and Flagella Cilia: short hairlike projections used in cellular movement; found in paramecium, oviducts. Flagella: whiplike projection used in cellular movement; found in algae, protozoa and some bacteria. Cilia are shorter than flagella. Both act as locomotor appendages of some cells (a) Motion of flagella. A Direction of swimming flagellum usually undulates, its snakelike motion driving a cell in the same direction as the axis of the flagellum. Propulsion of a human sperm cell is an example of flagellate locomotion. 1 µm (b) Motion of cilia. Cilia have a back-and-forth motion that moves the cell in a direction perpendicular to the axis of the cilium. A dense nap of cilia, beating at a rate of about 40 to 60 strokes a second, covers this Colpidium, a freshwater protozoan.  Cilia and flagella contains “9+2” tubule arrangement. At the base of cilia and flagella are basal bodies. Basal body has a structure familiar to the centriole (“9+0” pattern). Cilia & flagella: “9 + 2” pattern ring  Cytoskeleton A network of fibers extending throughout the cytoplasm. Functions: - mechanical support of cells - maintain the shape of cells - attachment of the organelles and enzymes - enable cell to change shape Cytoskeleton has 3 components: microtubules, actin filaments and intermediate filaments.  Microtubule 0.25 µm Microfilaments  Cell Wall Thick layer outside the cell membrane used to give a cell strength and rigidity Consist of a network of fibres, which give strength but are freely permeable to solutes (unlike membranes) Made mainly of cellulose, but can also contain hemicellulose, pectin, lignin and other polysaccharides Absent  Plasma Membrane The plasma membrane separates the internal environment of the cell from its surroundings. Cell and organelle membranes are composed of two layers - phospholipid bilayers. Fluid mosaic model by Singer and Nicolson. 1. Fluid Structure of Membranes Membranes are not static. Layers move over each other based on percent of unsaturated fatty acids. Most protein and phospholipid molecules can move laterally. lateral diffusion flip-flop rare rotation 2. Fluid Structure of Membranes Proteins and other molecules are embedded in a framework of phospholipids.  Structure of Cell Membrane Extracellular matrix Glycoprotein Carbohydrate Plasma membrane Glycolipid Microfilaments of cytoskeleton Integral Cholesterol Peripheral Cytoplasm protein protein Phospholipid  Extracellular matrix Oligosaccharide Glycoprotein Intrinsic protein Glycolipid Cholesterol Phospholipid bilayer Channel protein Cytoplasm Extrinsic protein  Cells live in fluid environments, with water inside and outside the cell. Hydrophilic (water-loving) polar heads of the phospholipid molecules lie on the outward-facing surfaces of the plasma membrane. Hydrophobic (water-fearing) nonpolar tails extend to the interior of the plasma membrane. Plasma membrane proteins may be peripheral proteins or integral proteins.  Aside from phospholipid, cholesterol is another lipid in animal plasma membranes; related steroids are found in plants. Cholesterol strengthens the plasma membrane. When phospholipids have carbohydrate chains attached, they are called glycolipids. When proteins have carbohydrate chains attached, they are called glycoproteins. Carbohydrate chains occur only on the exterior surface of the plasma membrane.  In animal cells, the carbohydrate chains of cell recognition proteins are collectively called the glycocalyx. The glycocalyx can function in cell-to-cell recognition, adhesion between cells, and reception of signal molecules. Membrane Proteins Proteins of the plasma membrane provide 6 membrane functions: 1. transport proteins 2. receptor proteins 3. enzymatic proteins 4. cell recognition proteins 5. attachment proteins 6. adhesion proteins Transport Proteins Channel proteins: channel for lipid insoluble molecules and ions to pass freely through. Carrier proteins: bind to a substance and carry it across membrane, change shape in process. Receptor Proteins It binds hormones and other substances on the outside of the cell. Binding triggers a change inside the cell called signal transduction. E.g. the binding of insulin to insulin receptors causes the cell to put glucose transport proteins into the membrane. Enzymatic Proteins Many membrane proteins are enzymes. Carry out enzymatic reactions right at the membrane when a substrate binds to the active site. Cell Recognition Proteins Glycoproteins and glycolipids identify type of cell and identify a cell as “self” versus foreign. The carbohydrate chains on membrane surface vary between species, individuals, and even between cell types in a given individual. Attachment Proteins Attach to cytoskeleton (to maintain cell shape and stabilize proteins) and/or the extracellular matrix. Adhesion Proteins Help cells stick together to form tissues. Play a key role in creating tight and anchoring junctions.