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  1. 1. Department of Natural Sciences University of St. La Salle
  3. 3. All living organisms are constructed from cells. Cells come in varied shapes and sizes
  4. 4. MICROSCOPY Interaction of probe used (photons: light, phase contrast, polarizing & fluorescence microscopy; electron beams: EM), and tissue components produce image Considerations in microscopic analysis: that the probe being utilized must not be larger than the detail to be seen that the probe and object being investigated must interact it must be possible to observe and interpret this interaction Units for measuring microscopic dimensions:
  6. 6. IMPORTANT TERMS IN MICROSCOPY Magnification – increases the apparent size of the specimen; a property of both ocular & objective lenses Working distance is the distance between the specimen and the magnifying lens. Depth of field is a measure of the amount of a specimen that can be in focus. Highly sensitive video cameras enhance power of microscopes, and create digitized images that can be fed into computers for quantitative image analysis
  7. 7. • Numerical aperture – a measure of the size or angle of the cone of light delivered by the illuminating condenser lens to the object plane and of the cone of light emerging from the object.• Resolving power – a measure of linear distance of the smallest degree of separation at which 2 details can still be distinguished from each other; dependent on quality of objective lens; R also varies according to the refractive index at the interface of the media used• Generally resolution increases with magnification, although there comes a point of diminishing returns where magnification is
  8. 8. PREPARATION OF TISSUES FOR MICROSCOPIC EXAMINATION (MICROTECHNIQUE)1.Fixation - prompt treatment of tissues in fixatives for about 12 hrs. (depending on tissue size) prevent autolysis by enzymes or bacteria, and preserve their morphologic and molecular composition.• To render the structural components insoluble, chemicals that precipitate the proteins are used.• The best fixatives are those that produce fine precipitates, e.g. buffered isotonic solution of 4% formaldehyde and glutaraldehyde react with amine groups (NH2) of proteins, or cross-link with protein
  9. 9.  Gross distortions without basis in the structure of the living cell are termed fixation artifacts. Examples of artifacts: • swelling and shrinkage of tissue components due to poor fixation, dehydration and/or embedding techniques; • wrinkles, tears, air bubbles due to poor sectioning technique; • dust and stain precipitate in section resulting from use of old stain solutions, use of improperly filtered or unfiltered stain solutions, mistakes made during preparation of the stain, or poor
  10. 10. Folded artifact Cracked tissue artifact Knife mark + folded artifact
  11. 11. 2.Dehydration and Clearing  Bathing of tissue in graded concentrations of organic solvents (70- 100% ethanol) to replace tissue water within 6-24 hrs.  Ethanol is then replaced with solvent miscible in embedding medium (xylene, benzene, toluene)  WHY? Most fixatives are water soluble, most embedding media are non-polar and are not miscible with water.  Dehydration moves the tissue from a polar (water-based) medium to a non-polar medium (e.g. toluene) that is miscible with the embedding medium.
  12. 12. 3.Embedding in melted paraffin at 58-600C, or plastic resin at room temperature  Tissue will be sectioned, and needs to be durable enough to withstand the sectioning process.  Embedding in wax or plastic immobilizes structural components of tissue. Holds them in place as sectioning is done.  Embedding medium must penetrate all cellular/intercellular spaces to impart rigid consistency to tissue before sectioning  Tissue shrinkage and artifacts may result from heat needed for paraffin embedding; virtually absent in resin embedding
  13. 13. 4.Sectioning by microtome to a thickness of 1-10 m Sections are then floated in warm water and transferred to glass slides Allows histologist to: see internal structure of tissue. stains or specific markers such as antibodies to more easily infiltrate the tissues. light to pass through tissue making structure visible.
  14. 14.  Images from thin sections are 2-D; living tissues are 3-D In order to understand the architecture of an organ, sections made in different planes should be studied How different 3-dimensional structures may appear when thin-sectioned. A: Different sections through a hollow ball and a hollow tube. B: A section through a single coiled tube may appear as sections of many separate tubes. C: Sections through a solid ball (above) and sections through a solid cylinder (below).
  15. 15. 5.Staining– to differentiate the colorless tissue elements as certain cellular elements take up more stain than others, producing a contrast that allows observation of structure not visible in unstained tissue. It may also reveal differences in chemical nature of regions of the tissue.6.Mounting – stained sections are placed on a slide in a gummy medium that hardens. The preparation is then covered with a thin
  16. 16. TYPES OF MICROSCOPES1.Light microscopes- compound, dissecting, brightfield, and phase-contrast Best resolution is 0.2 µm. Anton van Maximum Leeuwenhook magnifications are (1632-1723) between 1000X and 1250X.
  18. 18.  Compound microscopes bring small objects "closer" to the observer by increasing the magnification of the sample. Since the sample is the same distance from the viewer, a "virtual image" is formed as the light passes through the magnifying lenses.
  19. 19. Bright-field phase-contrastNomarski differential- phase-contrast dark-field Phase contrast microcopy- uses a lens system that changes light speed as it passes through structures with different refractive indices • The phase of the light is altered by its passage through the cell, and small phase differences can be made visible by exploiting interference effects • Phase-contrast and differential interference optics produce 3-D images of transparent living cells,
  20. 20. Contrast in Phase MicroscopyContrast in LightMicroscopy
  21. 21. 2.Fluorescence microscopy– uses strong UV light source that irradiate substances dyed with fluorescent stains, e.g. acridine-orange• These appear as brilliant, shiny particles on a dark background; useful for identifying & localizing NA in cells• Fluorescence spectros- copy analyzes light emitted by fluorescent compounds in a micro- spectrophotometer• This permits highly sensitive assays of cellular substances such as catecholamines
  22. 22. The confocalmicroscope produces opticalsections by excludingout-of-focus light
  23. 23. 3. Polarizing Compact bone microscopy– birefrigent substances rotate direction of polarized light emerging from polarizing filters• Useful for visualizing substances with repetitive, oriented Collagen fibers, molecular polarizing microscopy structures
  24. 24. 4.Electron microscopy– uses high energy electron beams (between 5,000 - 109 electron volts) focused through electromagnetic lenses. Interaction of electrons deflected by lenses beamed on tissue components permits high resolution (0.2 - 1 nm) and 400x greater magnification than light microscopes The increased resolution results from the shorter wavelength of the electron beam Disadvantages of EM: requirements of a vacuum-enclosed system, high voltage, mechanical stability; special treatment & sample preparation make it highly complex and costly; requires the services of well-trained personnel
  25. 25. TEM S E M
  26. 26. Scanning vs. Transmission EM:• In the TEM, the image is formed directly on the image plane.• In the SEM, the image is formed indirectly by accumulation of information from the specimen point by point.• There is no need to cut ultra thin sections because the beam of the SEM does not pass through the specimen.• The resolution of the SEM is about 100 Angstrom vs. 4-5 Angstrom achieved by the transmission type.• The SEM has great depth of field making it possible to obtain 3-D images.• TEM magnifications are commonly over 100,000X• SEM displays images on high resolution TV
  27. 27. SEM: T-lymphocyte, E.coli TEM: mitochondria &attacked by macrophage chloroplast
  28. 28. Specimen Preparation for EM:• Fixation in osmium tetroxide, osmium dichromate, acrolein and glutaldehyde.• Since registration of color is not possible with the EM system, staining with colored dyes is not done in EM studies.• Specimen is mounted on a copper grid covered with carbon and/or plastic film
  29. 29. Freeze-cleaving, Freeze- etching or Cryofracture methods• Used with EM; replicas are made of surfaces of frozen aqueous materials at very low temperatures in vacuo• The use of chemical fixatives, dehydrating and embedding agents are avoided by using a freezing microtome/cryostat which permit sections to be obtained without
  30. 30. • Freezing does not inactivate most enzymes, hinders diffusion of small molecules, eliminates dissolution of tissue lipids by solvents• The tissue is impregnated chloroplast thylakoid membranes with a 25% glycerol solution before rapid freezing in liquid nitrogen or Freon12 at 1000C to 1550C.• Not entirely free of artifacts; valuable in the study of membranes and vesicles their junctional
  31. 31. QUESTIONS?