Introduction to Methods of Morphology & Cell Biology INMD 6801Fall 2014Jan Norrander, Ph.D.Dept. Genetics, Cell Biology & DevelopmentUniversity of Minnesota!!!!!!!!!!!!!!!!!!!!!!!!!!!CellsMembrane organelles, cytoskeleton, molecules(Chapters 2 & 3)TissuesCells + extracellular matrixOrgansBasic tissue types interacting to form a Functional Morphological UnitOrgan SystemsOrgans working together to provide a basic physiological needCorrelation of Microscopic Structure to Function (Physiology/Molecular Biology)HistologyFig 12.18 Human Anatomy3/e (© Benjamin Cummings 2001) !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!By the End of This Week You Will Be Able to:1. Define basic optical properties. 2. Compare and contrast the different types of light & electron microscopy. 3. Describe how tissues are prepared for microscopy4. Be able to interpret the information from 2-dimensional images into 3-dimensional structure. 5. Use a light microscope to view cells and tissues. 6. Decipher electron microscopic images. 7. Recognize the principal cell organelles and components by light and electron microscopy.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!MicroscopesLight microscopy (LM)Interactions of lightwith tissue componentsElectron microscopy (EM)Interactions of electronswith tissue componentsProduce images, then enlarge the image to make small objects visible to the naked eye!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Using light to create an image!!!!Basic Concepts of Optical SystemsDetection vs ResolutionDetection: Ability to perceive the presence of an objectResolution: Discrimination between two objects that are close togetherEvery optical system has a finite resolving powerMagnifying objects beyond this resolution will be ‘empty magnification’Terminal Bars Junctional ComplexNucleiTerminalBars© Robert L. Sorenson!!!!!!!!!!!!!!!!!!!!!!!!!!Here can only see 1 object!They haven't been resolved!!!!!!!!!!!!!More intense stain= more proteins= probably a structure. EM have better resolving power.!They have been resolved hereResolutionResolving power of an optical system –minimum distance two objects must be separated in order to be seen as separateResolving power of some optical systems:Unaided eye 250 micronsLight microscope 0.25 micronsElectron microscope 0.001 micron (1 nm)(for biological specimens)!!!!!!!!!!!!!!!!!!!!Objects can be as close as 250 microns for our eyes and we can still tell they are two distinct objects. Closer together-> they start to look like one.!!!!!!!!!!!!!!!!!!!!Wavelength and ResolutionWavelength -distance between peaks of the waveformResolving power = 0.61 x N.A. = wavelength= 0.55 µm for LM= 0.004 nm for EM (100 kV)!!!!!!N.A. = numerical aperture !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Numerical Aperture (N.A.) and ResolutionN.A. = Numerical aperture~ ability of a lens to gather lightResolving power = 0.61 x N.A.Fig 9-6 Molecular Biology of the Cell 5/e (©Garland Science 2008)!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!The more light gathered by a lens, the more info it has to create an image, the better the image.MagnificationMagnification refers to the size of the image relative to the size of the objectPurpose of a microscope is to create an image of an object (e.g. a cell) and then magnify that image to the point where it is within the resolving power of our eyesSize of a RBC = 7 micronsLimit of resolution of unaided eye = 250 microns!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Magnification1X magnification40X magnification100X magnification400 X magnification!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Bringing those RBCs w/in resolving power of your eye.!!Magnification is Related to ResolutionThe higher the resolution, the more an image may be enlarged (magnified) and the more details you will be able to visualizeMicroscopes have several objective lenses, each with a different N.A. and image magnificationLimit of resolution = 0.61 x N.A.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!NA= ability to gather light.!Magnify 5x on the first one-> reach empty magnification (can't get more info from the image)!!!!!!!!!Greater Resolving Power of Electron Microscopes Allows Visualization of OrganellesFig 1-8 Essential Cell Biology 3/e (©Garland Science 2010)© R.L. SorensonMagnifying beyond the resolving power of a optical system results in ‘empty magnification’ (magnification without revealing more detail)!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!High resolution EM. Can see more detail.Types of MicroscopyLight microscopy (LM)Interactions of lightwith tissue componentsElectron microscopy (EM)Interactions of electronswith tissue componentsBrightfield – stained specimensPhase Contrast – unstained specimensNomarski/DIC – unstained specimensFluorescence – localization of proteinsLaser Scanning Confocal – produce 3D viewsTransmission EM (TEM)Scanning EM (SEM)Think of resolving power/resolution like digital images. Putting pic on billboard.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!lab->!!!!!!!!!!!!Light MicroscopeCondenserObjectivesEyepiece(Ocular)!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Condenser lens focuses the light through the specimen you have on the stage. !Multiply magnifying power by objective lens. Usually 10x.!What You Can SeeLightMicroscopy(Resolving power = 0.25 microns)ElectronMicroscopy(Resolving power = 0.001 microns)Sorenson, Atlas of Human HistologyFawcett, The Cell 2/e (© W.B. Saunders 1981)!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Preparation of Tissues for Light Microscopy• Fixation (preservation of biological structure)•Dehydration (alcohols)•Clearing (xylene)• Embedding (paraffin or plastic resin)• Sectioning (5 to 10 µm)•Sections mounted on microscope slides•De-waxing and Re-hydration• Staining (Detection Method)• Dehydration•Mounting Media applied•Coverslip appliedLiver© SorensonFig 9-10 Molecular Biology of the Cell 5/e (© Garland Science 2008)!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Contrast and DetectionTo detect an object, it must contrast with its surroundings.Most cells are colorless and translucent making them difficult to visualize.The phase of light is altered by its passage through either thicker or denser parts of a cell. By amplifying this effect, unstained cells can be made visible. Figure 9-7 Molecular Biology of the Cell 5/e (©Garland Science 2008)!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Alter phase of light by passing through thicker regions-> amplify phase diff-> visible cells!!!!!BrightfieldPhase contrast
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