Module 2 - Microscopy Flashcards
(21 cards)
Light Refraction
When light passes from one medium to another, it is refracted, or bent, at the interface
The refractive index is a measure of how greatly a material slows the light velocity
The direction and magnitude of the bending is determined by the refractive indices of the two media forming the interface
Lenses
Lenses focus light rays at a specific point: the focal point
the focal point is the distance between center of lens and the focal point
The strength of a lens related to its focal length
the shorter the focal length, the greater the magnification
Microscope Resolution
the resolution is the ability of a lens to separate or distinguish small objects that are close together
For light microscopes, the wavelength of light used is major factor in resolution
-> the shorter wavelenght the greater resolution
Abbe equation
d=0.5(λ)/nsinθ
d= resolving power, min distance between 2 objects to distinguish them
nsinθ = numerical aperture (ability to gather light)
n is refractive index of medium
θ is amount of light entering lens
λ is wavelength
-> for best resolution: smallest d->smallest λ largest nsinθ
Light microscopes
there are many types of light microscopes
bright field
darkfield
phase contrast
fluorescence
Light microscopes are compound microscopes, where the image is enlarged by the action of 2 or more lenses
Two lens system
objective is nearer the specimen
-magnifies the specimen
-designated by focal length
-shorter the focal length, shorter the working distance
Eye-piece is the ocular
-produces virtual image by the eye
Total magnification = ocular magnification x objective magnification
Sample preparation - staining
increases visibility and/or accentuates specific morphological features
Sample preparation - fixing
preserves internal and external structures (organisms killed and firmly attached to microscope slide)
-heat fixation - routinely used with prokaryotes (preserves overall morphology, but not internal structures)
-chemical fixation - used with larger, more delicate organisms (protects fine cellular substructures and morphology)
Dyes/Staining
increases the contrast with the backgroung
improves the visualization of internal and external structures
Can stain for specific structures (flagella, endospores, etc)
Dyes have two common features
-Chromophore groups (chemical groups with conjugated double bonds that give the colour)
-Ability to bind with cells (ionic,covalent,hydrophobic bonding)
Simple staining
single stain is used
simple and easy to use
useful in determining size,shape and arrangement of cells
dyes that bind by ionic interactions are among the most common
-Basic dyes - bind to negatively charged molecules (DNA, proteins, Cell surface)
-Acidic dyes - bind to positively charged cell structures
Differential staining
used to differentiate organisms based on their staining properties
Gram-staining
-most widely used differential stain
-Differentiates based on differences in the cell wall structure
-Acid-fast staining
Bright-Field Microscope
produces a dark image against a brighter background
Has several objective lenses
Total magnification is the product of the magnification of the ocular lenses and the objective lenses
Dark-field
Image is formed by light reflected or refracted by specimen
-Produces a bright image of the object against a dark background
Used to observe living, unstained preparations
For eukaryotes, has been used to observe internal structures
Phase contrast
Enhances the contrast between intracellular structures having slight differences in refractive index
Excellent way to observe living cells
-Especially useful for detecting bacterial components such as endospores and inclusion bodies that have refractive indices from that of water
Fluorescence
The specimens are usually
stained with one or more
fluorescent dyes called
fluorochromes
* The labeled sample is
exposed to ultraviolet, violet,
or blue light
* A bright image is formed from
the emitted fluorescent light
Electron Microscope
Light microscopes have a
resolution limit of ~ 0.2 m
* In electron microscopy, images
are produced by beams of
electrons
* The wavelength of the electron
beam is much shorter than that of
light
– Significantly higher resolution
– Very useful for detailed studies of
many microorganisms
– Necessary for studies of viruses
Transmission Electron Microscopy
Electrons scatter when they
pass through thin sections of
a specimen
* Transmitted electrons
(those that do not scatter)
are used to produce image
* The denser regions scatter
more electrons and appear
darker
TEM sample prep
- Fixed with glutaraldehyde
- Dehydrated with acetone or alcohol
- Added to liquid epoxy to permeate cell, then
hardened (polymerized) - Thin sections cut with microtome (glass or diamond
blade) - Cells made “electron dense” with heavy metal salts
(eg. lead or uranium) - The stained thin sections mounted on copper grids
and visualized
Scanning electron Microscope
- Uses electrons reflected
from the surface to create
the image - Produces a 3-D
visualization of the surface - Samples must be fixed,
dried, and coated in metal
confocal Microscopy
- Confocal scanning laser
microscope - Samples are frequently
fluorescently labeled - Live cell imaging is possible
- Laser beam is used to illuminate
a variety of planes in the
specimen
– Computer compiles the images
created from each point to generate
a 3-D image
Scanning Probe Microscopy
- Scanning tunneling microscope
– Steady tunneling current maintained between the
microscope probe and the specimen
– Up and down movement of probe as it maintains
the constant current is detected and used to an
create image of surface of specimen - Atomic force microscope
– Sharp probe moves over the surface of the
specimen at constant distance
– Up and down movement of probe as it maintains
constant distance is detected and used to create
an image
