MICROSCOPY Flashcards
Small pieces of tissue are placed in solutions of chemicals that cross-link proteins and inactivate degradative enzymes, which preserve cell and tissue structure.
FIXATION
The tissue is transferred through a series of increasingly concentrated alcohol solutions, ending in 100%, which removes all water
DEHYDRATION
Alcohol is removed in organic solvents in which both alcohol and paraffin are miscible.
CLEARING
The paraffin-infiltrated tissue is placed in a small mold with melted paraffin and allowed to harden.
EMBEDDING
The tissue is then placed in melted paraffin until it becomes completely infiltrated with this substance.
INFILTRATION
The resulting paraffin block is trimmed to expose the tissue for sectioning (slicing) on a microtome.
TRIMMING
The hardened block with tissue and surrounding embed- ding medium is trimmed and placed for sectioning in an instrument called a
MICROTOME
TYPES OF LIGHT MICROSCOPY
Bright-field, Phase-contrast, Differential interference contrast microscopy, Dark-field, Fluorescence, Confocal
What is the most commonly used staining method?
Combination of the stains H&E which act as basic and acidic dyes
Cell substances with a net negative (anionic) charge, such as DNA and RNA, react strongly with hematoxylin and basic stains; such material is said to be..
BASOPHILIC
Cationic substances, such as collagen and many cytoplasmic proTeins react with eosin and other acidic stains and are said to be..
ACIDOPHILIC
This microscope is most commonly used by both students and pathologists, uses o ordinary light and the colors are imparted by tissue staining.
BRIGHT-FIELD MICROSCOPY
This microscope uses UV light, under which only fluo- rescent molecules are visible, allowing localization of fluorescent probes which can be much more specific than routine stains.
Fluorescence microscope
This microscope uses the differences in refractive index of various natural cell and tissue components to produce an image without staining, allowing observation of living cells.
Phase-contrast microscope
This microscope involves scanning the specimen at succes- sive focal planes with a focused light beam, often from a laser, and produces a 3D reconstruction from the images.
Confocal Microscopy
This process localizes cell components synthesized using radioactive precursors by detecting silver grains produced by weakly emitted radia- tion in a photographic emulsion coating the tissue section or cells.
Autodiography
This technique use specific enzymatic activities in lightly fixed or unfixed tissue sections to produce visible products in the specific enzyme locations.
Enzyme Histochemistry or Histochemical techniques
Specific place where light rays are focused
Focus point
distance between center of lens and focal point
Focal length
(T/F) Short focal length = more magnification
TRUE
(T/F) Shorter wavelength = greater resolution
TRUE
Distance between the front surface of lens and surface of cover glass or specimen when it is in sharp focus
Working Distance
- Image is formed by light reflected or refracted by the specimen
- Produces a bright image of the object against a dark background
- Used to observe living, unstained preparations
- Used to observe internal structures in eukaryotic microorganisms
- Very thin histological sections can be used if unstained or only certain components are stained, as in silver stains
Dark field microscope
● Exposes specimen to ultraviolet, violet, or blue light
● Specimens usually stained with fluorochromes
● Shows a bright image of the object resulting from the fluorescent light emitted by the specimen
● Has applications in medical microbiology and microbial ecology studies
● For viewing
The fluoresence microscope
● This creates sharp, composite 3D image of specimens by using a laser beam, aperture to eliminate stray light, and
computer interface
● Numerous applications including the study of biofilms
● Laser
● For demonstrating specimens
Confocal Microscope
● use 20,000 - 120,000 volts of beams of electrons to create highly magnified images
● Electrons replace light as the ‘illuminating’ beam that will pass through glass lens
● Wavelength of electron beam is much shorter than light, resulting in much higher resolution and magnification
● Allows for study of microbial morphology in great detail
Electron Microscope
2 types of electron microscope
TEM & SEM
● Electrons scatter when they pass through thin sections of a specimen
● Transmitted electrons are under vacuum which reduces scatter and are used to produce clear image
● Denser regions in specimen scatter more electrons and appear darker
● Produces a 2D image
TRANSMISSION ELECTRON MICROSCOPE
○ A type of staining that make internal and external structures of cell more visible by increasing contrast with background
DYES
(T/F) ○ Basic dyes have positive charges
○ Acid dyes have negative charges
TRUE
