microscopy

• Histology


  Study of tissues
• Histology


  • Study of normal tissues
• Levels of Organization


  • Atoms
  • Molecules
  • Cells
  • Tissues
  • Organs
  • Organ system
  • Organism
• Histologist


  Performs microscopy to study the structure of normal tissues
• Histopathologist


  Performs microscopy to study the structure of abnormal tissues
• We need to study the appearance of normal tissue to gain a thorough understanding of it
• Cytology


  Study of structure and function of the plant and animal cell
• Organology


  Study of organ and organ system
• Chondrocytes


  • Responsible for cartilage formation
  • Found inside the cartilage itself
• Types of microscopes


  • Brightfield microscope
  • Darkfield microscope
  • Phase-contrast microscope
  • Fluorescent microscope
  • Electron microscope
• Brightfield microscope


  • Contains two lens systems for magnifying specimens (ocular lens in the eyepiece and objective lens in the nosepiece)
  • Specimen is illuminated by a beam of tungsten light
  • Specimen appears dark against a bright background
  • Specimens are usually nonviable, stained preparations
  • Direct light
  • Limitation - absence of contrast between the specimen and the surrounding medium, making it difficult to observe living cells
  • Light source: tungsten-halogen bulb (3200k) warm light
  • Most routinely used
• Darkfield microscope


  • Similar to the ordinary light microscope
  • The condenser system is modified so the specimen is not illuminated directly
  • The condenser directs the light obliquely so the light is deflected or scattered from the specimen
  • The specimen appears bright against dark background
  • Living specimen may be observed more readily
• Phase Contrast Microscope


  • Observation of unstained microorganisms is possible
  • Includes special objectives and a condenser that makes visible cellular components that differ slightly in their refractive index (bending of light rays)
  • The image appears dark against light background
• Fluorescent Microscope


  • Used most frequently to visualize specimens that are chemically tagged with a fluorescent dye
  • The source of illumination is an ultraviolet (UV) light (0 – 400nm)
  • Light source: High pressure mercury lamp or hydrogen quarts lamp
  • The ocular lens is fitted with a filter that permits the longer ultraviolet wavelengths to pass, while the shorter wavelengths are blocked or eliminated
  • UV radiations are absorbed by the fluorescent label and the energy re-emitted in the form of a different wavelength in the visible light range
  • fluorescent dyes absorb at wavelengths between 230 – 350 nm and emit an orange, yellow or greenish light
• Fluorescent dyes


  • Acridine orange dye
  • DAPI stain
• Acridine orange dye


  • N: cell nuclei
  • R: RNA rich cytoplasm
• DAPI stain


  • green: acting filaments
  • blue: nuclei
• Fluorescent Microscope


  • used primarily for the detection of antigen-antibody reactions
  • antibodies are conjugated with a fluorescent dye that becomes excited in the presence of UV light
  • the fluorescent portion of the dye becomes visible against a black background
• Electron Microscope (EM)


  • Provides a revolutionary method of microscopy
  • Magnifies up to one million
  • Permits visualization of submicroscopic cellular particles and virus
  • The specimen is illuminated by a beam of electrons rather than light
  • Focusing is carried out by electromagnets instead of a set of optics
  • Components are sealed in a tube in wherein a complete vacuum is established
  • Specimens should be thinly prepared, fixed and dehydrated to allow the electron beam to pass through freely
• Types of Electron Microscope


  • Transmission EM
  • Scanning EM
• Transmission EM


  • Requires specimen that are thinly prepared, fixed, and dehydrated to allow the electron beam to pass through freely
  • As the electrons pass through the specimen, images are formed by directing the electrons onto the photographic film- that makes the internal cellular structure visible
  • Resolution: 400,000X in detail
  • Magnification: 120,00X
• Scanning EM


  • Used for visualizing surface characteristics rather than intracellular structures
  • A narrow beam of electrons scans back and forth, producing a three-dimensional image as the electrons are reflected off the specimen's surface
  • Dried, then spray a thin layer of heavy metal (gold)
• Parts of a Compound Brightfield Microscope


  • Body tube
  • Arm
  • Base
  • Mechanical stage
  • Stage clips
  • Ocular or eyepiece lens
  • Illuminator or light source
  • Condenser
  • Diaphragm
  • Revolving nosepiece or turret
• Body tube


  Connects the eyepiece to the objective lens
• Arm


  Supports the body tube and connects it to the base
• Base


  Bottom of the microscope and used for support
• Mechanical stage


  • Fixed platform with an opening in the center to allow for the passage of light from an illuminating source below to the lens system above the stage
  • Provides a surface for the placement of a slide with its specimen over the central opening
  • Can be moved vertically or horizontally by means of adjustment
• Stage clips


  Holds the slides in place
• Ocular or eyepiece lens


  • Lens at the top of the microscope
  • Usually 10 – 15x
• Illuminator or light source


  • Positioned in the base of the instrument
  • Built in light source- provides direct illumination
  • Mirror (one side flat and other concave) – indirect illumination
  • Uses an external light source (lamp or sunlight) – placed in front of the mirror to direct the light upward into the lens system
  • Flat side: artificial light
  • Concave side: sunlight
• Microscope care - Once the microscope is on the table


  1. Remove all unnecessary materials

  2. Uncoil the microscope's electric wire and plug into the outlet

  3. Clean all lens systems - ocular and objective

  4. Use methanol or xylol (xylene) - lens cleanser
• Condenser


  • Found directly under the stage and contains two sets of lenses that collects and concentrate light passing upward
  • Illuminates the object with rays of light that pass through the specimen obliquely as well as directly
• Microscope care - After using the microscope

  1. Clean all the lenses with a dry, clean lens paper with a drop or two of methanol

  2. Use xylol to clean the mechanical stage

  3. Place the objective back to LPO

  4. Center the mechanical stage

  5. Coil the electric wire around the body tube

  6. Return the microscope
• Diaphragm


  • Rotating disk under the mechanical stage
  • Can be adjusted to change the intensity and size of the cone of light that is projected upwards of the slide
  • Used to create contrast on cellular components
  • Tissues – open diaphragm more
  • Cells or urinary sediments – close for better contrast
• Revolving nosepiece or turret


  • Holds the objective lenses
  • Allows for easy rotation of objective lenses
• Objective Lenses


  • Scanner objective (4x)
  • Low Power Objective (10x)
  • High Power Objective (40x)
  • Oil Immersion Objective (100x)
• Scanner objective (4x)


  • Red color
  • Shortest objective
  • Useful for getting an overview of the slide
  • For whole organs like section of the spinal cord, lung, digestive tract
  • Safe to use since it cannot be lowered to the point of contacting with the slide
• Low Power Objective (10x)


  • The most useful lens for viewing slides
  • Almost any feature can be featured in this objective
  • Also safe to use since it cannot be lowered to the point of contacting with the slide
• High Power Objective (40x)


  • Also called as high dry objective
  • Used for observing fine detail such as striations in skeletal muscle, types of nerve cells in the retina, etc.
  • Usually, cells are being tested
• Oil Immersion Objective (100x)


  • The longest objective
  • Used for observing details of the individual cells such as WBC, RBC and immature cells
  • Must be together used with immersion oil