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Transcript
Introduction to the Microscope Care Parts Focusing • • • • • Always carry with 2 hands Only use lens paper for cleaning Do not force knobs Always store covered Keep objects clear of desk and cords Eyepiece Body Tube Revolving Nosepiece Objective Lens Stage Clips Diaphragm Light Arm Stage Coarse Focus Fine Focus Base Light Microscopes • Place the Slide on the Microscope • Use Stage Clips • Click Nosepiece to the lowest (shortest) setting • Look into the Eyepiece • Use the Coarse Focus • Follow steps to focus using low power • Click the nosepiece to the longest objective • Do NOT use the Coarse Focusing Knob • Use the Fine Focus Knob to bring the slide What can you find on your slide? Microscope One or more lense that makes an enlarged image of an object. What microscopes do - designed for: 1. Magnification: the relative enlargement of the specimen when viewed through the microscope 2 Pictures of a flea at same magnification. (a) was acquired using optics that provided higher resolution than (b) 2. 3. Resolution: the ability to discern fine details. The resolution of a microscope is determined by the wavelength of light (or energy) used for illumination. For light microscopy,the limit is ~200 nm. Contrast: the difference in intensity between the image and the background. Contrast is produced in the specimen by staining with colored dyes that absorb light, by using special optical techniques, or by using fluorescent probes. Microscopy • Microscopes are instruments designed to produce magnified visual or photographic images of small objects. The microscope must accomplish three tasks 1. produce a magnified image of the specimen 2. separate the details in the image, 3. render the details visible to the human eye or camera. • • • • Simple Compound Stereoscopic Electron Simple Microscope • Similar to a magnifying glass and has only one lense. Compound Microscope • Lets light pass through an object and then through two or more lenses. Stereoscopic Microscope • Gives a three dimensional view of an object. (Examples: insects and leaves) Electron Microscope • Uses a magnetic field to bend beams of electrons; instead of using lenses to bend beams of light. Electron Microscopes • The use of high energy electrons to examine the fine details of objects. Major Types of electron microscopes. • Scanning electron microscope (SEM). • Transmission electron microscope (TEM) Scanning electron microscopy (SEM). Types of specimens: -Whole organisms -Natural tissue surfaces -Exposed tissue structure -Corrosion casts SEM of Penicillium islandicum Transmission electron microscopy (TEM). • Allows the observation of molecules within cells • Allows the magnification of objects in the order of 100, 000’s. Representative EM images of Pst DC3000 avrPto (pAVRPTO), immunogold-labeled with the AvrPto antibody. In situ immunogold labeling was done after bacteria were grown in hrp-inducing medium for 4 hours, supplemented with (A) no SA, (B) SA for 4 hours, or (C and D) SA for 1 hour. Dark dots are 15-nm gold particles in (A) and (C) and 10-nm gold particles in (B) and (D). Arrows indicate Hrp pili attached to rodshaped bacteria (only a portion of the bacterium is shown, surrounded by dark stain). Scale bars, 100 nm. A Lense • Enlarges an image and bends the light toward your eye. Eyepiece Lense Usually has a power of 10 x Eyepiece Lense X Objective Lense = Total Magnification Low Power = 4 x Medium Power = 10 x High Power = 40 x A Tour of the Cell Topics • • • • • Microscopy Cell Fractionation Eukaryotic Cells Prokaryotic cells Organelles Microscopy • Magnification – Ratio of object’s image to real size • Resolution – Measure of clarity of image – Minimum distance two points can be separated • Light Microscopy – 1590 – Improved in 17th century Microscopy • Electron Microscope: – 1950 – Focuses beam of electrons through the specimen or on the surface – Achieve resolution up to 0.002nm – Biological structures need resolution 2nm • Scanning Electron Microscope • Transmission Electron Microscope Microscopy • Scanning Electron Microscope – Detailed study of surface of specimen – Surface is coated with gold film – Beam excites electrons – Electrons are captured by a device which translates the pattern in an electronic signal to a video screen. – Image appears three dimensional Microscopy • Transmission Electron Microscope – Electronic beam through a thin section of specimen – Certain cellular structures get stained with atoms of heavy metals – Electron density specific to certain parts of cells – Instead of glass lenses, electromagnets are used to bend electrons – Photographic images are viewed – Sometimes digital camera is attached Microscopy • Many organelles detailed structure revealed • Light microscopy preferred over electron microscopy when studying live cells • Methods used to prepare specimen kills the cells • Microscopes are widely used in cytology • Cytology combined with biochemistry Figure 7.1 The size range of cells Cell Fractionation • Fractionation is done by centrifugation • Centrifugal force separates cell components based on size and density • Ultracentrifuge can spin 130,000 rpm and 1 million times force of gravity • Different time span and g value can yield different cell organelles • Cytologists use microscopy to reveal the different organelles in a pellet • Biochemists use biochemical methods to determine metabolic functions Figure 7.3 Cell fractionation Cells • Eukaryotic cells – Protists, fungi, animal and plant cells • Prokaryotic cells – Bacteria and Archaea • Difference • Plant cell and Animal cell – Structure – Difference Microscope Resolution • ability of a lens to separate or distinguish small objects that are close together • wavelength of light used is major factor in resolution shorter wavelength greater resolution Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 38 Preparation and Staining of Specimens • increases visibility of specimen • accentuates specific morphological features • preserves specimens Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 39 Fixation • process by which internal and external structures are preserved and fixed in position • process by which organism is killed and firmly attached to microscope slide – heat fixing • preserves overall morphology but not internal structures – chemical fixing Copyright © The McGraw-Hill Companies, Inc. Permission required fo reproduction or display. • protects fine cellular substructure and morphology of larger, more delicate organisms 40 Dyes and Simple Staining • dyes – make internal and external structures of cell more visible by increasing contrast with background – have two common features • chromophore groups – chemical groups with conjugated double bonds – give dye its color • ability to bind cells Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 41 Dyes and Simple Staining • simple staining – a single staining agent is used – basic dyes are frequently used • dyes with positive charges • e.g., crystal violet Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 42 Figure 7.7 Overview of an animal cell Figure 7.8 Overview of a plant cell Cells Plant cell Animal Cell Chloroplast Lysosomes Central vacuole and tonoplast Centrioles Cell wall Flagella Plasmodesmata Structures • Nucleus: – Most genes in eukaryotic cells – Some genes also present inside chloroplast and mitochondria – Covered with nuclear envelope – DNA is organized inside the nucleus in the form of chromosomes – Chromosome is bound to form chromatin – Chromosome number is specific for different species Structures • Nucleus: – Nucleolus – Densely stained granules and fibers next to chromatin – Special type of RNA (rRNA) synthesis – Proteins imported from cytoplasm are assembled with rRNA – Sometimes there are two or more nucleoli – mRNA is transcribed in the nucleus and is transported out through nuclear pores Structures • Ribosomes: – Cells with high rate of protein synthesis have more ribosomes – Free ribosomes are suspended in cytoplasm – Bound ribosomes are attached outside of ER – Most proteins formed in cytosol on free ribosomes function in the cytosol – Most bound proteins work as membrane proteins or transport proteins Figure 7.10 Ribosomes Structures • Ribosomes: – Cells with high rate of protein synthesis have more ribosomes – Free ribosomes are suspended in cytoplasm – Bound ribosomes are attached outside of ER – Most proteins formed in cytosol on free ribosomes function in the cytosol – Most bound proteins work as membrane proteins or transport proteins Figure 7.11 Endoplasmic reticulum (ER) Structures • Endoplasmic Reticulum: – Network of membranous tubules and sacs called cisternae – Continuous with nuclear envelope – Smooth ER • Synthesis of lipids • Metabolism of carbohydrates • Detoxification of drugs & poisons • Enzymes of smooth ER synthesize lipids, oils, phospholipids, steroids • Very active in testes and ovaries Structures • Endoplasmic Reticulum: – Smooth ER • Detoxification process in liver cells • Addition of hydroxyl group • Stores calcium ions in muscle cells • Calcium ions released from smooth ER can trigger different responses – Rough ER • Protein Synthesis • Secretory proteins • Glycoproteins (by specialized molecules on ER) • Transport Vesicles: Vesicles in transit Structures • Golgi Apparatus – Transport vesicles travel to Golgi apparatus – Products of ER are modified and stored – Arranged in stack with membranous sac called cisternae – Membranes at ends differ in thickness & composition – Cis and trans face (correspond to receiving and shipping ends) Structures • Golgi Apparatus – Transport vehicles travel to Golgi apparatus – Center of manufacturing, warehousing, sorting, and shipping area – Products of ER are modified and stored – Arranged in stack with membranous sac called cisternae – Membranes at ends differ in thickness & composition – Cis and trans face (correspond to receiving and shipping ends) – Cis is closer to ER Figure 7.12 The Golgi apparatus Structures • Golgi Apparatus – Manufactures certain macromolecules – Many polysaccharides like pectins, noncellulose polysaccharides made by plant cells – Cisternae move forward making golgi apparatus dynamic Structures • Lysosomes – – – – – – – – Digestive compartments Sac of hydrolytic enzymes Work in acidic environment Hydrolytic enzymes and lysosomal membrane are made by ER Transferred to golgi apparatus Proteins present inside the lysosomal membrane and digestive enzymes are protected Lysosomes carry out intracellular digestion Lysosomes use their enzymes to recycle cell’s own materials (autophagy) Structures • Vacuoles – Plant or fungal cell specific – Similar to lysosomes – Food Vacuoles – Contractile vacuole-> Pumps excess of water out of cell – Central Vacuole Mature Plants • Enclosed by tonoplast • Tonoplast is selective • Reserves of the plant Structures – Central Vacuole Mature Plants • Storage for inorganic compounds (potassium, chloride) • Disposal site for metabolic sites • Contain pigments • Can store poisonous substances • Growth of plant cells Structures • Mitochondria – Energy transformer – Site of cellular respiration – Generates ATP’s by extracting energy from sugars, fats and other fuels – Not part of endomembrane system – Two layered membrane – Membrane proteins made by free ribosomes – Contain small amount of DNA – Semiautonomous organelles Structures • Mitochondria – Some cells may have single large mitochondrion or several – 1-10 micrometer long – Outer membrane smooth – Inner membrane - cristae – The mitochondrial matrix has enzymes and the DNA Structures • Chloroplast – Found in plant cells and algae – Convert solar energy to chemical energy – Not part of endomembrane system – Two layered membrane – Membrane proteins made by free ribosomes – Contain small amount of DNA Structures • Chloroplast – Specialized member of family “plastids” • Amyloplasts: Colorless plastids storing starch like roots and tubers • Chromoplasts: Pigments that give color to fruits and flowers (orange and yellow) • Chloroplast: Have green pigment chlorophyll – Interconnected stacks called “thylakoids” – Each stack is called “granum” – Fluid outside the thylakoids is called “stroma” – Chloroplast DNA and other enzymes present Structures • Peroxisomes – Specialized metabolic compartment – Contain enzymes that help transferring various substrates to Oxygen – Produces Hydrogen peroxide as a result – Several roles • Break fatty acid by using oxygen • In liver detoxifies alcohol by giving hydrogen to oxygen • Hydrogen peroxide is then broken to water by an enzyme • Specialized peroxisomes: Glyoxysomes Structures • Cytoskeleton – Network of fibers extending the cytoplasm – 3 types of Molecular structures • Microtubules • Microfilaments • Intermediate filaments Structures • Extracellular Components – Cell Wall: • Protects plant cell, maintains the shape , and prevents excessive uptake of water • Microfibrils made of polysaccharide cellulose are embedded in the matrix • Young plant makes “primary cell wall” • Between primary wall of adjacent cell is “middle lamella” • Layer rich in pectin (polysaccharide) • Glue like substance used in jams and jellies • Either by hardening or adding secondary cell wall Structures • Extracellular matrix – Main ingredient glycoprotein – Collagen – Collagen fibers in animals are embedded in network of proteoglycans – Some cells are bound to ECM by “fibronectin” – These all bind to surface receptor proteins called “integrins” Structures • Intercellular Junctions – Plasmodesmata: Plant cell walls have channels – Cytosol passes through them and maintains interconnectivity between cells – Water and small solutes along with RNA molecules and certain proteins can pass from cell to cell Structures • Intercellular Junctions • In animals (epithelial cells) – Tight Junctions: • Membranes of neighboring cells are tightly pressed – Desosomes • Function like anchors/rivets • Fastening cells • Keratin proteins help to anchor – Gap Junctions • Cytoplasmic channels from one cell to next cell • Special membrane proteins present