生物代寫 - CEDB20003 Fundamental of cell biology
ALC1 Visualising Cells??CEDB20003 2020ALC1 VisualisingCells??????CEDB200032020Learning task1. Define resolution and factors that affect resolutionResolution(D) is the minimum distance between two objects that enables them to be distinguished astwo separate objects.Lambda is the wavelength of lightn is the refractive index of the medium through which the light is passingTheta is the angular aperture or half the width of the cone of rays collected at the midpoint of theobjective lensThe higher the magnification of the objective, the lager the angular aperture.The higher the refractive index(n), the higher the resolutionResolution is impacted by the refractive index of the medium (usually either air, water or oil) throughwhich the light is passing from above the specimen to the objective lens.2. Describe the key principles and limitations of light microscopyEyepiece DO NOTincreasetheresolution
The condenser lens improves the illumination of the specimen.The objective lens resolves details in the specimen. The numerical aperture of this lens determines itsresolution.The objective and the eyepiece lenses combine to magnify the image to a size greater than the limit ofresolution of the human eyeThelimitsofresolutionofthelightmicroscopeisabout0.2umor200nm,wecan see bacteria,mitochondria and cell muckiest, maybe large vesicles and granules. But we cannot see virus orsmaller intracellular organeles such as ribosomes.Transmisssion electron microscopesUltra-thin tissue sections are treated with heavy metals that deflect electrons.Electrons are fired onto the specimen and the image is detected from transmitted electrons.Structures that bind metals the most (nucleolus, vesicles) show as dark areas.Structures that do not bind metals (cytoplasm, vacuoles) show as light areasSimilarities and differences between the principal features of a light microscope and a transmissionlectron microscope
The ense in the light microscope are made of glass, those in the electron microscope are mageneticcoils.The electron microscopes requires that the specimen be placed in a vacuum.Scanning electron microscopeUsed to image surface structure.The specimen is coated with heavy metals and an image is generated from back-scattered electrons(not transmitted electrons)the specimen is scanned by a beam of electrons brought to a focus on the specimen by theelectromagnetic coils that act as lenses.The detector measures the quantity of electrons scattered or emitted as the beam bombards eachsuccessive point on the surface of the specimen and controls the intensity of successive points in animage built up on a screen.The SEM creates striking images of three-dimensional objects with great depth of focus and aresolution between 3 nm and 20 nm depending on the instrument.Recognise and compare SEM and TEM imagesSEM - Scanning Electron MicroscopeUltra-thin tissue sections are treated with heavy metals that deflect electrons.Electrons are fired onto the specimen and the image is detected from transmitted electrons.Structures that bind metals the most (nucleolus, vesicles) show as dark areas.
Structures that do not bind metals (cytoplasm, vacuoles) show as light areasTEM - Transmission Electron MicrocsopeUsed to image surface structure.The specimen is coated with heavy metals and an image is generated from back-scattered electrons(not transmitted electrons)Describe the role of thickness in microscopy and methods to visualise thick objectsThick objects must be sectioned when using transmitted radiation. Making tisssue sectionsFor reference, a sheet of paper is about 100 μm thick.For light microscopy we require sections ~1 - 50 μm thick.For electron microscopy we require sections ~50 - 100 nm thickProblems with sectioningHistological processing: Chenical fixation, dehydration and embeddingRapid freezingThese processesKilling living things
Can cause changes in cell structure - artefactsThree-dimensional shape must be recostructed - singal thin sections sometimes give misleadingimpression. The true three-dimensional shape can be reconstructed from a complete set of serialsection.What is contrast?Contrast is the relative difference in brightness/darkness or colour between an object and itssurroundings, or between different parts of an objectDescribe the role of contrast in microscopy and methods to increase contrastLiving cells are usually transparentlack of contrastMethod to increase contrastOptical Methods:enhance microscope imagesStaining:thinsectionsoffixedtissueSelectivelabeling: The most widely used example is Green Fluorescent Protein (GFP)Theroleoflight phase of contrast insight microscopyLight passing through the unstained living cell experiences very little change in amplitude, and thestructural details cannot be seen if the image is highly magnified.The phase of the light, however, is altered by its passage through either thicker or denser parts of thecell, and small phase differences can be made visible by exploiting interference effects using a phase-contrast or a differential-interference-contrast microscopeDiffereftialInterferenceCotrast(DIC)microscopyUsingwithoutstainingcanincreasecontrastCanobservecellintheirnature stateTheobjectiveneedstobethinsingle-celled organisms and small, transparent multicellular organisms or embryos;individual cells which can be extracted and spread out on a slide, e.g. blood smear, cervical smear,analysis of semen (sperm count and motility)
ThreetypedoflightmicroscopyBrightfield-light is transmitted straight through the specimen.Phase contrast - phase alterations of light transmitted through the specimen are translated intobrightness changes.DIC-which highlights edges where there is a steep change of refractive index.Light waves passing through parts of an object that differ in thickness and/or refractive index willbecome out of phase, as shown in the diagram above. Phase contrast and DIC microscopy convertthese phase differences into differences in light intensityDisadvantage-Lacksepicificitythey cannot be used to highlight just one part of the cell or just onetype of cell in a tissue. Furthermore, they cannot be used to locate where a particular class ofmolecules is located in a cell or tissue.StainingWhenwestaintissue,weareable to abserve diffferent colours due to different stains absorbingspecific wavelength of light.StainingtoincressecontrastinlightmicroscopyWillabsorblightofsomewavelengthH&EclassicstainingmethodHaematoxylin, a blue/purple dye that binds to negatively charged (i.e. acidic or basophilic, whichmeans 'base-loving') molecules. This includes nucleic acids (DNA, RNA), so haematoxylin stains thenucleus and parts of the cell rich in ribosomes.
Eosin, a pink dye that binds to positively charged (i.e. basic or acidophilic, which means 'acid-loving')molecules. This includes many proteins and therefore eosin stains cell cytoplasm, collagen andmuscle. Red blood cells are almost all cytoplasm and therefore stain uniformly pink with eosinCompare methods of examining live cells/tissues versus fixed cells/tissuesDemonstrate basic light microscopy skills to visualise cell and tissue structureFluorescent dyesExcited by light at one wavelength and emit light at longer wavelengthFluorescence microscopyProvide very high contrast images . This enable objects to be visualise that contain only a smallnumber of dye molecules. Thus fluorescent dyes are more sensitive.The fluorochrme emit light of a particular wavelength while also ensuring that litttle or no light fromunlabeled reons is seen.How does fluorescence work?Excitation - cause by a specific narrow band of wavelengthEmission - a different definite colour always have a longer wavelengthGreen Fluorescent Protein (GFP)Naturally occur protein with blue lightThe gee coding of GFP can be attache to the gene coding of any protein normally made by cellGFP- labeled proteins can simply be visualized using fluorescence microscopyNon-toxic to cellDoes not affect the function of the protein which it attachDescribe the key principles of fluorescent microscopyOptical filters are used to select an appropriate wavelength to excite the fluorescent dye in useEnsure that only the wavelength of light emitted by the dye pass to the viewer. This achieve the highcontrast typical of fluorescence microscopy.epi-illumination- The separation of the excitation and emission wavelengths that is the goal offluorescence microscopy is best achieved if the excitation light shines on the specimen from above
Fluorescence immunohistochemistryUse for staining specific cells, parts of cells or molecules in cellsThe techniques use antibodies o perform specific labeling (antibodies are promade by the immunesystem in vertebrates, they enable the body’s defenses to recognize foreign proteins in invading virus,bacteris or other microorganism - these protein targets are antigens.)Casting the protein we wish to investigate as the antigen.Inject the antibody in protein into the mammal.Apply a solution of this ntibody to the cells we are studyingThe antibodies will bind tightly only to the antige if and where it is present.A fluorescence microscopy is used to visualise where the antibody is bound in the cell or tissue.Direct VS indirect immunohistochemistryDirect immunohistochemistry - chemically couple the fluorescent label to the antibody molecule (theprimary antibody) that recognises the antigen of interest. ( not very convenient because a fluorescenttag has to be added to each and every type of primary antibody that a researcher wishes to use.)Indirect immunohistochemistr- the primary antibody does not carry the fluorescent marker. It marker on another type of antibody,the secondary antibody which recognise binds to the primary antibody. ( very sensitive )Demonstrate basic light microscopy skills to visualise cell and tissue structureResolution and magnificationWhat is resolution? Write a definitionand then discuss this with the rest of your group.What is the resolving power of a typical young person with normal vision?
Look closely at the skin on one of your fingers. What must the resolution of your optical instrumentbe, approximately, if you wish to:Determine the arrangement of cells in skin?Identify organelles in skin cells? e.g. differentiate Golgi body from mitochondria.Visualise details of the internal structure of organelles of skin cells? e.g. examine what is inside amitochondrion.Build a microscope activity??
Practical microscopy skillsHow thick or thin is the tissue section?Is it translucent or opaque?Are the colours you see in the sections natural?Are the cells in the tissue still alive, or at least fairly fresh?Is there any aspect of the tongue tissue you can more readily see with the naked eye than with amicroscope?More generally, in what situations would it be worthwhile looking at a microscope slide with thenaked eye before examining the slide under the microscope?What does trans-illumination mean?Why does this method of illumination require the tissue section to be relatively thin?How do you calculate the overall magnification obtained?What is the role of the condenser?