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.
Direct v. Indirect Immunocytochemistry
Uses labeled antibodies to provide precise
localization of molecules
– Develop an antibody against the macromolecule of
interest
– Label the antibody with a fluorescent dye
• Two methods: direct and indirect
– Direct = antibody against the macromolecule is labeled with a fluorescent dye
– Indirect = antibody against the antibody against the
macromolecule is labeled with a fluorescent dye
Indirect is more sensitive because multiple anti-antibodies can bind to the primary antibody
Phase Contrast Microscope
• A light microscope used to visualize
unstained cells and tissues
- cells can still be living!!
• Uses the fact that light changes speed when
passing through structures with different refractive
indices
Differential Interference Contrast Microscope
Similar to phase contrast
Additional prisms to rotate light
Images appear 3-D
Polarization Microscope
• Specimen between two polarizing filters
• Structures made of oriented molecules (e.g. collagen) that rotate the light
– Bright structures against dark background
– Birefringence = ability to rotate the direction or
vibration of polarized light
Used to study things like crystals or highly organized molecules of collagen – these types of molecules rotate the light and you can use this type of microscope to collect the light that is rotated in a certain direction
Because this microscope collects light wavelengths that are going in a specific direction, it is BLOCKING any other wavelength and so, the background is BLACK!!
Confocal Microscope
• Only a very thin plane of the specimen is in focus
– Beam of light is very narrow
– Image passes through a pinhole
In a conventional microscopy, you are actually seeing multiple layers of specimen in focus; but with this microscope, you are only viewing ONE of those planes
Light bounces off the specimen and the pinhole in the detector allows only one plane beam of light to pass and blcks the rest! allowing for a really focused image
Confocal Microscope Illumination
• Illumination is by laser scanned over specimen
• Possible to combine several focused planes into
one 3-D image
• Use fluorescent molecules to label structures
Electron Microscope
Two Types:
- Transmission - Scanning electron
Use this microscope if something is too small to see with a light microscope
It uses the same principles as bright field but using electrons as opposed to light; so instead of glass lenses that refract light, we are using electromagnetic lenses that bend the beams of electrons
Light source is on top and specimen is at the bottom (opposed of light microscope)
Imaging detected by fluorescent screen (TEM), electron detector (SEM), or photographic plates
Need to use different staining and embedding techniques
Can only view dead/insert objects
Electron micrographs are in black and white:
- Dark areas = electron dense; electrons blocked - Light areas = electron lucent; passes through
Stains that block electrons are used (heavy metals like
uranium or gold)
SEM vs. TEM
• Transmission electron microscopy – electron
beam passes through a very thin (40-90 nm) section
(internal part of cell)
• Scanning electron microscopy – electron beam is
scanned across the specimen; electrons react with a metal coating that was applied to the specimen and reflected or emimed electrons are detected
(surface of cells)
Freeze Fracture
1) Freeze specimen
2) Fracture specimen
Fracture will occur along natural lines of weakness like plasma membrane
3) Coat with carbon and platinum
4) View with EM
