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DEFINITION OF TERMS:
• Resolving power/Resolution • Working Distance • Magnification • Numerical Aperture
• Refractive Index
• Capacity of the microscope to
separate clearly 2 points
• Determined by the shortest wavelength of visible light and maximum numerical aperture
Working Distance • Distance between the surface of the lens and the surface of the cover glass or the specimen when it is in sharp focus
• Objectives with large NA and great resolving power have short working distanceworking distances
Magnification • Ratio of image size to the actual size • Objective Magnification X Ocular Magnification .
the higher the magnification . n=1) and the angle of light (u) made by the lens NA= n X sin u • The larger the NA.Numerical Aperture • Measure of the size or angle of the cone of light entering the aperture of the lens • Light-gathering capacity of the microscope • A number that indicates the resolving power of a lens system • Derived mathematically from the refractive index(n) of the optical medium (for air.
Refractive Index • Measure of the light-bending ability of the medium such as glass or air • Light bends as it passes from the glass slide into the air. immersion oil placed between the glass slide and the OIO lens prevents the refraction or scattering of light rays that would otherwise be lost to the objective lens . bending is due to the fact that the glass slide and air have different refractive indices • Immersion oil has the same refractive index as the glass.
gives general outline HPO.01 40 mm 10X 0. examines microorganisms .35 um 0.5-0. locates various structure LPO. lower magnification.25 16 mm 40-45X 0. higher magnification.55-0.8-2. yellow ring. largest lens. lowest magnification.shorter. gives details of specimen OIO. smaller lens.18 um power with light of 450 nm Scanner-shortest objective.longest.longer.The Properties of the Microscope Objectives property magnification NA scanner LPO HPO OIO 4X 0. highest magnification. white ring. larger lens.9 um 0. small lens.1 mm Approximate resolving 2.3 um 0. red ring.7 mm 0.65 4 mm 90-100X 90-100 1. for initial focusing. blue ring.0 mm Approximate focal length Working distance 17-20 mm 4-8 mm 0.
such as micrometers and nanometers • Micrometer (um)= 10-6 m • Nanometer (nm)= 10-9 m • Angstrom (A)= 10-10 m or 0. has the advantage of easy conversion by a single factor of 10 • The standard unit of length is the meter (m) • Microorganisms and their structural components are measured in even smaller units.Units of Measure • When measuring microorganisms. we use the metric system.1 nm .
TYPES OF MICROSCOPE • Light or Optical Microscope a. fluorescence e. dark-field f. differential interference c. phase-contrast d. SEM . bright-field • UV Microscope • Electron Microscope a. polarizing microscope b. TEM b.
2 um • Brightfield illumination is used for stained smears. Resolution: 0.000X.The Light Microscopes • Uses visible light as a source of illumination • Specimen appears against a dark or bright background • Magnification: 2. most common is the binocular compound microscope • Unstained cells are observed using modified compound microscopes .
The Nature of Light Waves How are waves quantified? Crest and trough Amplitude . measured in seconds. This is known as P.maximum excursion from its undisturbed or relaxed position.the distance a wave travels during one complete oscillation. and is recorded in units of Hertz. Period . v. The number of crests that pass at a specific point in space is called a wave’s frequency or f. f = 1/P Wavelength() = speed x P or speed/f .the time it takes for one complete cycle. Wavelength . Waves travel at a speed.
the rays interact to produce reinforcement (relative brightness) However.MECHANISMS OF LIGHT MICROSCOPE The principle is based on the wave nature of light rays. If the wave peak of light rays from one source coincides with the wave peak of light rays from another source. and the fact that light rays can be in phase (their peaks and valleys match) or out of phase. the rays interact to produce interference (relative darkness) . if the wave peak from one light source coincides with the wave through from another light source.
Brightfield Microscope imaging of S. aureus .
POLARIZING MICROSCOPE • Birefringence : Capacity to change the direction of the axis of light • Modification : with two filters POLAROID : between the light source and condenser ANALYZER : at the draw tube • Applications : mineral elements ash residues spindle fiber .
A POLARIZING MICROSCOPE .
the meteorite Tieschitz needles of urates from gout .
focusing light on the specimen and a diffraction plate in the objective lens.are brought together. different protoplasmic constituents produce phase variations One set of light rays comes directly from the light source. they form an image on the ocular lens.direct rays and reflected or diffracted rays. containing areas that are relatively light (in phase). direct and reflected or diffracted light rays are brought together to produce the image Principle: Combination of in-phase and out-of-phase. the diaphragm allows direct light to pass through the condenser. the other set comes from light that is reflected or diffracted from a particular structure in the specimen • • • When the 2 sets of light rays.PHASE-CONTRAST • Distinguishing features: uses a special condenser containing an annular (ringshaped) diaphragm. through shades of gray. to black (out of phase) Application : facilitates detailed examination of the internal structures of living specimens • .
uses 2 light beams separated by beamplitting prisms.DIFFERENTIAL INTERFERENCE • Like phase contrast. adding contrasting colors to the specimen • Provides a colored 3D image . uses differences in refractive indexes to produce images • However.
Differential Interference Contrast Microscope .
anthracis (appears apple green) .FLUORESCENCE MICROSCOPE • Takes advantage of fluorescence.g. tuberculosis (glows yellow) fluorescein isothiocyanate (FITC) for B. the ability to absorb light at a shorter wl (ultraviolet) and emit it at a longer wl (visible) • Uses an UV or near-UV source of illumination that causes specimen to emit light or fluoresce. The specimen appears luminescent bright object against a dark background • the specimen can either be fluorescing in its natural f orm or stained with fluorochromes e. fluorochrome auramine for M.
FLUORESCENCE MICROSCOPE cont’d Parts: LIGHT SOURCE : Hg vapor lamp : Quartz iodine lamp OPTICAL SYSTEM : 2 barrier filters 1 dichroic beamsplitting mirror Application: for fluorescent-antibody techniques (immunofluorescence) to rapidly detect and identify microbes in tissues or clinical specimens .
FLUORESCENCENCE MICROSCOPE .
Fluorescence Microscope Configuration .
or are distorted by staining.DARKFIELD MICROSCOPE • Uses a special condenser with an OPAQUE DISC that blocks light from entering the objective lens directly • Light reflected by the specimen enters the objective lens. frequently used to detect T. do not stain easily. and the specimen appears light against a bright background • APPLICATION : examines living microorganisms that are invisible in brightfield microscopy. pallidum in the diagnosis of syphilis .
objective lens stage condenser lens DARKFIELD MICROSCOPE CONFIGURATION .
red blood cells under dark field microscope .
CONFOCAL MICROSCOPE • A computerized microscope that uses laser light to illuminate one plane of a specimen at a time • Images are taken point-by-point and reconstructed with a computer • Application: obtains 2.or 3-dimensional images of cells for biomedical application .
CONFOCAL MICROSCOPE CONFIGURATION .
maps atomic and molecular shapes 2.SCANNED-PROBE MICROSCOPE • Uses probes to examine surface of a specimen at a very close range without causing modification or damage • Application: 1. characterizes magnetic and chemical properties 3. determines temperature .
produces a 3D image .resolution is greater than that of an EM 2. Atomic Force Microscope (AFM) .uses a metal-and-diamond probe .provides incredibly detailed views of molecules such as DNA .SCANNED-PROBE MICROSCOPE cont’d Types: 1.uses a thin metal (tungsten) probe .no special preparation is required . Scanning Tunneling Microscope (STM) .
Detector SCANNED PROBE MICROSCOPE CONFIGURATION .
flagellae of Pseudomonas putida .
fluorescence of special glasses. in a scanning instrument the receptor is a multiplier phototube.UV MICROSCOPE utilizes quartz. . fluorite. and other ultraviolettransmitting lenses the image is made visible by photography. or television.
UV MICROSCOPE .
ELECTRON MICROSCOPE • Ultrastructure and subcellular structures • UNIQUE FEATURES : e.beams (W filaments) fluorescent screen/TV monitor electromagnetic field .
high magnification • DISADVANTAGES : Requires vacuum enclosed system high voltage mechanical stability different way of specimen preparation well-trained staff .ELECTRON MICROSCOPE • ADVANTAGES: high resolution.
Two Types of ELECTRON MICROSCOPE .
filament condenser lens condenser aperture stage objective lens objective aperture projector lenses .