Tools of Basic Med. Flashcards
Four basic types of Tissues
– Epithelium
– Connective Tissue
– Muscle
– Nervous Tissue
Light Microscope
• Based on the interaction of tissue components
with light
uses visible light - light on the visible spectrum to interact with tissues in different ways
• Different types: bright field, phase contrast,
differential interference, polarizing, confocal,
fluorescence, etc.
• Simplest technique is bright field microscopy
How a Light Microscope works
Light source
• Series of lenses
– Condenser: collects and focuses light
– Objective: lenses magnify the image of the object
and project it towards the eyepiece
– Eyepiece: further magnifies the image and projects it
toward the eye or a camera
– Magnification is calculated by multplying the
magnifying power of all lenses
– Maximum magnification is approx. 1000-1500X
• Stage holding specimen slide
Resolution
• Resolution = the smallest distance between two
particles at which they can be seen as separate
objects
• Resolution is determined by wavelength of
illumination and by numerical aperture (NA)
• Maximal resolving power of light microscope is 0.2
μm
SO basically, resolution allow us to see objects as separate from one another
Resolution Formula
R = 0.61 λ/NA
• R = resolution = minimal resolvable distance between
distinguishable points
• λ = wavelength of the illumination source
• NA = numerical aperture = n sin(μ)
– n = refractive index of the medium between the lens and the specimen (1.00 for air, 1.51 for oil)
– μ = angle of light cone (< 90°)
Minimize R by:
(1) decreasing the wavelength of the light source (λ)
and/or by
(2) increasing the NA by
(a) increasing the refractive index of the medium
and/or
(b) increasing the angle between the stage and
objective.
Preparation of Tissues
- Fixation – to preserve the structure and
molecular composition
• Formaldehyde or glutaraldehyde
• Cross linkage of macromolecules - Decalcification – removal of calcium (if necessary) to section (Put in acid solution to leech out minerals)
- Dehydration – removal of water
• Graded series of ethanol (50% to 100%) - Clearing – replace alcohol with a solvent that can
form a solution with the embedding medium
• Xylene - Embedding – to provide rigidity in order to
facilitate secConing
• Melted paraffin, hardens at room temperature - Sectioning – cut into very thin (1-10 μm) slices
• Microtome - Rehydra-on – replace water (most stains are water-soluble)
- Staining
Measurements
1 μm = 1 micron = 1 micrometer = 1 millionth of a
meter = 0.001 mm = 1000 nm
A red blood cell is 6-8 μm in diameter and are often used as a good way to estimate size in a slide because their size is fairly standard
In light microscopes, things will be usually measured in microns, but electron microscopes are usually in nanometers.
Staining
• Types of stains
– Stains that differentiate between acidic and basic
components of the cell
– Specialized stains that differentiate the fibrous
components of the extracellular matrix
– Metallic salts that form metal deposits on tissues
• Common stain is H&E
– Hematoxylin = stains acidic components of a cell
(DNA, RNA, ribosomes, etc.) blue
– Eosin = acidic dye that stains basic components of
a cell (cytoplasm) pink
Basophilic
cell components that stain with a basic dye; so it stains ACIDIC COMPOUNDS
– Methylene blue, toluidine blue, hematoxylin
Acidophilic
cell components that stain with an acidic dye; so it stains BASIC COMPOUNDS
– Acid fuchsin, orange G, eosin
Metachromatic
a tissue or cell component that stains a different color than the dye
– Dye molecules form aggregates in the presence
of polyanions in the tissue; aggregates differ in
color from individual molecules
EXAMPLE:
Toluidine blue stains non-metachromatic tissue blue,
but stains granules of mast cells purple
Periodic acid-Schiff (PAS)
used to stain carbohydrate-rich molecules
– Polysaccharides (e.g. glycogen), glycoproteins,
glycolipids, mucins
think PAS-ta; pasta is high carbs!!
Silver
- Structures that stain with silver are called argentophilic or argyrophilic
- Reticular fibers (type III collagen)
Tetracycline
Tetracycline will bind to bone during the mineralization process
Bone: fluorescent labeling with tetracycline
Tetracycline was given (to a living person) on two occasions, several days apart.
Can determine if mineralization was occurring during one or both of the labeling periods.
example: two lines indicated the tetracycline was being taken up both times - so mineralization occurred twice; only one line shows that it was being actively mineralized only on one occasion
CAUSES SIGNIFICANT STAINING ON TEETH
Fluorescent Staining
when certain substances are irradiated by light of a certain wavelength, they emit light with a longer wavelength
– Sections are irradiated with UV light or laser, and the
emission is in the visible portion of the spectrum.
– Fluorescent substances appear brilliant or colored on a dark background.
