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HISTOLOGY REVIEWER
Pointers to Review MICROSCOPY AND HISTOTECH
Microscopy and Histotech
Microscope
Greek, Micron=small. Greek, Scopos= aim instrument for viewing objects that are too small to be seen by naked eye or unaided eye Microscopy science of investigating small objects using such instrument is called “ Microscopy ” HISTORICAL BACKGROUND 1590 - Hans Janssen and his son Zaccharias Janssen developed first microscope 1609-Galileo Galilei - compound microscope 1620 - Christiaan Huygens , another Dutchman , developed a simple 2- lens ocular system that was chromatically corrected Anton Van Leeuwenhoek credited Brought the microscope to the attention of biologists ”A tradesman of Delft, Holland 1661 Anton Leeuwenhoek discovered bacteria , free living and parasitic
microscopic protists, sperm cells, blood cells, microscopic nematodes etc. *Microscope used by Anton Van Leeuwenhoek:
VARIABLES USED IN MICROSCOPY
Magnification
degree of enlargement no of times the length, diameter of an object is multiplied Resolution Ability to reveal closely adjacent structures details as separate and distinct Limit of Resolution min. distance between two visible bodies at which they can be seen as separate and not in contact with each other TYPE OF MICROSCOPE
LIMIT OF
RESOLUTION
Compound Microscope 0.2 micrometers Scanning Electron Microscope 0.020 micrometers Transmission Electron Microscope .0025 micrometers TYPES OF MICROSCOPE Simple Microscope
single lens , similar to magnifying glass provides low magnification used for simple observations like examining small objects such as insects or fibers Compound microscope multiple lens, (eyepiece and objective) Provides higher magnification Observes small details of cells and tissues, commonly used in laboratories and classrooms for biological studies
Illumination - Lamp, sunlight, battery operated lamp, 60 W Bulb, Quartz
halogen light METHODS OF USING COMPOUND MICROSCOPE
- Grasp the microscopes arm with one hand and place your other hand under the base
- Place the microscope on a bench. Adjust seat
- Clean Lenses
- Turn the coarse adjustment knob to raise the body tube
- Revolve the nose piece to set low- power objective lens
- Adjust the Condenser Lenses and Diaphragm
- Place a slide on the stage and secure with stage clips
- Switch on the light at low intensity and then increase intensity
- Turn the coarse adjustment knob to lower the body tube until the low power objective reaches its lowest point
- Looking through the eyepiece, very slowly move the coarse adjustment knob until the specimen comes into focus
- Adjust distance between eye piece
- Switch to high power objective lens only after adjusting condenser and iris diaphragm
- Place a drop of oil over specimen before using oil immersion objective
- Lower the objective lens until oil makes contact with objective
- Looking through the eyepiece, very slowly focus the objective away from the slide i.e by raising the objective lens. HOW TO OBSERVE A SLIDE
Slide should be scanned in an orderly fashion so that vital information is not missed **PHASE CONTRAST MICROSCOPE ** First described in 1934 by Dutch Physicist Frits Zernike Produces high- contrast images of transparent images Advantage- Living Cells can be examined in their natural state
ELECTRON MICROSCOPE
Uses a beam of highly energetic electrons to examine objects on a very fine scale. This examination can yield the info about
- Morphology
- Composition TYPES OF E.M
Transmission Electron Microscope(TEM) Scanning Electron Microscope(SEM) TRANSMISSION ELECTRON MICROSCOPE Stream of electrons is formed Accelerated using a positive electrical potential Focused by a metallic aperture and electromagnets Interactions occur inside the irradiated sample which are detected and transformed into an image Projector Lens forms image on Fluorescent viewing screen 2D Image Magnification 10,000X to 100,000X
SCANNING ELECTRON MICROSCOPE
Scan to give a 3d view of the surface of the object which is black and white used to study surfaces features of cells and viruses Scanning Electron microscope has resolution 1000 times better than Light microscope SEM IMAGES Treponema Pallidum Vibrio Cholerae with polar flagella
BASIC HISTOLOGICAL TECHNIQUES
- Fixation
- Tissues are mechanically and biochemically stabilized in a fixative
- The most common fixative is buffered isotonic solution of 4% formaldehyde
2)Dehydration and Cleaning
- Samples are immersed in multiple baths of progressively more concentrated ethanol to dehydrate the tissue, followed by a clearing agent such as, xylene or Histoclear
3)Embedding
- During this 12 to 16 hours process, paraffin wax will replace the water: soft, moist tissues are turned into a hard paraffin block, which is then placed in a mould containing more molten wax (embedded) and allowed to be cool and harden
- Embedding can also be accomplished using frozen, non fixed tissue in a freezing medium
- This freezing medium is liquid at room temperature but when cooled will solidify
- Sectioning
- Tissue is then sectioned into very thin (2-8 micrometer)sections using a microtome
- These slices, usually thinner than the average cell, are then placed on a glass slide for staining
5)Staining
- See the tissue under a microscope, the sections are stained with one or more pigments
- This is done to give contrast to the tissue being examined, as without staining it is very difficult to see difference in cell morphology - Hematoxylin and eosin (abbreviated H&E) are the most commonly used stains in Histology and histopathology - Hematoxylin colors nuclei blue, eosin colors the cytoplasm pink
- Other compounds used to color tissue sections include safranin, oil red, congo red, fast green, silver salts and numerous natural and artificial dyes Renal Cell Carcinoma
