ALC1 Visualising Cells CEDB20003 2020 ALC1 Visualising Cells CEDB20003 2020 Learning task 1. Define resolution and factors that affect resolution Resolution(D) is the minimum distance between two objects that enables them to be distinguished as two separate objects. Lambda is the wavelength of light n is the refractive index of the medium through which the light is passing Theta is the angular aperture or half the width of the cone of rays collected at the midpoint of the objective lens The higher the magnification of the objective, the lager the angular aperture. The higher the refractive index(n), the higher the resolution Resolution is impacted by the refractive index of the medium (usually either air, water or oil) through which the light is passing from above the specimen to the objective lens. 2. Describe the key principles and limitations of light microscopy Eyepiece DO NOT increase the resolution The condenser lens improves the illumination of the specimen. The objective lens resolves details in the specimen. The numerical aperture of this lens determines its resolution. The objective and the eyepiece lenses combine to magnify the image to a size greater than the limit of resolution of the human eye The limits of resolution of the light microscope is about 0.2 um or 200nm, we can see bacteria, mitochondria and cell muckiest, maybe large vesicles and granules. But we cannot see virus or smaller intracellular organeles such as ribosomes. Transmisssion electron microscopes Ultra-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 areas Similarities and differences between the principal features of a light microscope and a transmission lectron microscope The ense in the light microscope are made of glass, those in the electron microscope are magenetic coils. The electron microscopes requires that the specimen be placed in a vacuum. Scanning electron microscope Used 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 the electromagnetic coils that act as lenses. The detector measures the quantity of electrons scattered or emitted as the beam bombards each successive point on the surface of the specimen and controls the intensity of successive points in an image built up on a screen. The SEM creates striking images of three-dimensional objects with great depth of focus and a resolution between 3 nm and 20 nm depending on the instrument. Recognise and compare SEM and TEM images SEM – Scanning Electron Microscope Ultra-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 areas TEM – Transmission Electron Microcsope Used 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 objects Thick objects must be sectioned when using transmitted radiation. Making tisssue sections For 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 thick Problems with sectioning Histological processing: Chenical fixation, dehydration and embedding Rapid freezing These processes Killing living things Can cause changes in cell structure – artefacts Three-dimensional shape must be recostructed – singal thin sections sometimes give misleading impression. The true three-dimensional shape can be reconstructed from a complete set of serial section. What is contrast? Contrast is the relative difference in brightness/darkness or colour between an object and its surroundings, or between different parts of an object Describe the role of contrast in microscopy and methods to increase contrast Living cells are usually transparent lack of contrast Method to increase contrast Optical Methods: enhance microscope images Staining: thin sections of fixed tissue Selective labeling: The most widely used example is Green Fluorescent Protein (GFP) The role of light phase of contrast insight microscopy Light passing through the unstained living cell experiences very little change in amplitude, and the structural 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 the cell, and small phase differences can be made visible by exploiting interference effects using a phase- contrast or a differential-interference-contrast microscope Differeftial Interference Cotrast (DIC) microscopy Using without staining can increase contrast Can observe cell in their nature state The objective needs to be thin single-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) Three typed of light microscopy Brightfield- light is transmitted straight through the specimen. Phase contrast – phase alterations of light transmitted through the specimen are translated into brightness 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 will become out of phase, as shown in the diagram above. Phase contrast and DIC microscopy convert these phase differences into differences in light intensity Disadvantage – Lack sepicificity they cannot be used to highlight just one part of the cell or just one type of cell in a tissue. Furthermore, they cannot be used to locate where a particular class of molecules is located in a cell or tissue. Staining When we stain tissue, we are able to abserve diffferent colours due to different stains absorbing specific wavelength of light. Staining to incresse contrast in light microscopy Will absorb light of some wavelength H&E classic staining method Haematoxylin, a blue/purple dye that binds to negatively charged (i.e. acidic or basophilic, which means ‘base-loving’) molecules. This includes nucleic acids (DNA, RNA), so haematoxylin stains the nucleus 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 and muscle. Red blood cells are almost all cytoplasm and therefore stain uniformly pink with eosin Compare methods of examining live cells/tissues versus fixed cells/tissues Demonstrate basic light microscopy skills to visualise cell and tissue structure Fluorescent dyes Excited by light at one wavelength and emit light at longer wavelength Fluorescence microscopy Provide very high contrast images . This enable objects to be visualise that contain only a small number 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 from unlabeled reons is seen. How does fluorescence work? Excitation – cause by a specific narrow band of wavelength Emission – a different definite colour always have a longer wavelength Green Fluorescent Protein (GFP) Naturally occur protein with blue light The gee coding of GFP can be attache to the gene coding of any protein normally made by cell GFP
– labeled proteins can simply be visualized using fluorescence microscopy Non-toxic to cell Does not affect the function of the protein which it attach Describe the key principles of fluorescent microscopy Optical filters are used to select an appropriate wavelength to excite the fluorescent dye in use Ensure that only the wavelength of light emitted by the dye pass to the viewer. This achieve the high contrast typical of fluorescence microscopy. epi-illumination- The separation of the excitation and emission wavelengths that is the goal of fluorescence microscopy is best achieved if the excitation light shines on the specimen from above Fluorescence immunohistochemistry Use for staining specific cells, parts of cells or molecules in cells The techniques use antibodies o perform specific labeling (antibodies are promade by the immune system 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 studying The 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 immunohistochemistry Direct immunohistochemistry – chemically couple the fluorescent label to the antibody molecule (the primary antibody) that recognises the antigen of interest. ( not very convenient because a fluorescent tag 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 structure Resolution and magnification What is resolution? Write a definition and 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 instrument be, 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 a mitochondrion. Build a microscope activity Practical microscopy skills How 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 a microscope? More generally, in what situations would it be worthwhile looking at a microscope slide with the naked 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? Why is the condenser position adjustable? What is the working distance of an objective? For what reason would you need a reasonably long working distance? What sort of information can you obtain with the 4x objective? What extra information about the structure of cells and the tissue can you obtain with the 40× objective that was not seen at lower power? What sorts of things (organelles) can’t you see well even at this level of magnification? For example, can you see the cell membranes between two adjacent cells? Does using a 100× objective help? What are the relative advantages of low- and high-power magnification? Low-powerHigh-power Magnification e.g. 4x e.g. 40x Working distance Field of view Readily distinguish individual cells? List some likely uses or situations Define resolution (again) Has your understanding of resolution changed since you wrote a definition of resolution above? If so, write a more comprehensive definition below. How has your definition changed? Summarise the key factors that determine optical resolution in microscopy Explain why the highest power objective on your microscope has a much closer working distance than the low-power lens. Now, using your sense of scale, give an example of the smallest biological object that can be resolved with the 40x objective. Using the formula provided, calculate the resolution of the 40x objective on your microscope? What form of microscopy can go beyond the limitations of light microscopy? Summarise why H&E is a useful stain for interpreting this example of breast cancer histopathology. Can you think of an example of a question that would require specific labelling in living cells to get an answer? Can we expect to obtain better resolution with a fluorescence microscope than a microscope that uses transmitted light? Why? 2020 1
