Leccture 3 Flashcards
1
Q
Fundamentals of Light Microscopy
A
*Bright field microscopy most commonly used
*Typically employs compound microscope
*Common components:
*Light source
*Condenser lens
*Stage
*Objective lens
*Ocular lens
*Focusing knob
2
Q
Fundamentals of Light Microscopy
A
- Light passes through condenser lens, focused on specimen
- Light passing through specimen passes through objective lens,
parallel light rays now diverge - Divergent light rays pass through objective lens, further divergence
occurs - Divergence of once parallel light rays results in
magnification – increase in size of specimen - Total magnification – product of magnifying
power of objective and ocular lens
3
Q
Magnification
A
- Not the rate limiting factor (by far!)
- Magnification can be infinite
- At some point, no more information can be gathered
- Have reached the limiting factor of microscopy - RESOLUTION
4
Q
Resolution
A
- Minimum distance at which two objects can be distinguished
- Several factors affect resolution
- Wavelength of light used
- Refractive index of medium between lens and specimen
- Distance between lens and specimen
- Contrast
l = wavelength of light
h = refractive index
q = angle between most
divergent light ray gathered by
lens and the center of the lens
5
Q
Wavelength of Light Used
A
- Different colors of light have different wavelengths
*The shorter the wavelength, the higher the resolution - Many light microscopes use filters to select color of light
- What color of light in the visible spectrum would
provide the highest resolution?
6
Q
Refractive Index of Medium
Between Specimen and Lens
A
- Refractive index - ability to bend light
- Glass from the slide bends light more than air
- Light is bent away from lens as it passes through specimen
- Immersion oil has similar refractive index as glass
- More light is gathered by the lens, more information
7
Q
The Distance Between the
Lens and the Specimen
A
q = angle between most
divergent light ray
gathered by lens and the
center of the lens
The closer the lens to the specimen - the greater the value of q
8
Q
The Distance Between the
Lens and the Specimen cont
A
- Object is to obtain information
- In microscopy, light is information
- The further the lens from the
sample, the more light is lost - The closer the lense, more light
gathered
*More light means more information
and higher resolution
9
Q
Bright-Field Microscopy
A
- Condenser creates a
bright white
background against
which to see
specimens - Cells and organelles
within them are often
times transparent - Need a way to produce
contrast
10
Q
Preparing Cells for Staining
A
- Specimen must be very thin, thin sectioning for tissues, smears for
bacteria - Specimen must dry, allows for fixation
- Fixation attaches specimen to slide, preserves structure, multiple
ways to accomplish, heat or methanol common fixation techniques - Most simple stains – basic stains, (+) charge, binds to (-) charged
cell, electrostatic interaction - Net result, increases contrast
11
Q
Differential Staining – the Gram Stain
A
- Specimen prepared as for simple staining, 1 stain added – crystal violet
- Mordant added – iodine, causes crystal violet to form large aggregates – CVI
- Cells decolorized with alcohol
- Gm (+) cells retain CVI, Gm (-) cells decolorized
- 2 stain (counter stain) allows visualization of Gm (-) cells, safranin
- Gm (+) cells appear purple, Gm (-) cells appear red, provides information on structure
12
Q
Structure of the Gm (+) Cell Wall
A
- Outermost layer – peptidoglycan, composed of sugar and protein
- Alternating N-acetylglucosamine and N-acetylmuramic acid joined in
long chains by b-1,4 linkage - Form long chains that surround the cell
- Chains linked together by peptide crosslink, type of peptide depends
on species
13
Q
Structure of the Gm (+) Cell Wall cont
A
- Teichoic acids embedded in peptidoglycan layer
- Polyalchols composed of repeating residues of glycerol
phosphate or ribotol phosphate - Usually have sugars and D-Alanine attached to hydroxyl
groups of alcohol, always have phosphate ester group
attached - Negative charge allows binding of divalent cations (Ca++ and
Mg++) - Believed to have role in uptake of ions bound
14
Q
Structure of the Gm (-) Cell Wall
A
- Outermost structure is the outer membrane – lipid bilayer
- External surface of membrane contains unusual lipid – lipopolysaccharide – bacterial
endotoxin - Two components – polysaccharide chain and lipid A
- 2 domains for polysaccharide chain
- Core polysaccharide – conserved across species,
attaches chain to phosphate on lipid a through
amine ester linkage - Species specific O-antigen, variable external
region
15
Q
Structure of the Gm (-) Cell Wall cont
A
- Lipid A region of LPS comprises outer layer of outer membrane
- Bacterial endotoxin, uses NAG instead of glycerol
- Responsible for symptoms associated with Gm (-) infections (fever, malaise, etc.) – more
on this later - Interior layer of outer membrane comprised of “typical” lipids
- Peptidoglycan layer beneath outer
membrane, much thinner than Gm (+)
layer, 1-2 sheets thick - Plasma membrane beneath
peptidoglycan, space between outer
and inner membrane referred to as
periplasm
16
Q
Why does the Gram Stain Work?
A
- Several theories, I’ll tell ya my favorite one
- Crystal violet is charged
- Outer membrane of Gm (-) has hydrophobic core
- Crystal violet never comes into contact with peptidoglycan
- Must in order for iodine mordant to exhibit affect
- Therefore, gm (-) bacteria are distained and must be counterstained
- Color following Gm staining allows you to infer the structure of the
cell wall
17
Q
Other Types of Microscopy
A
- Bright field microscopy useful but limited
- Obtain information regarding structure of cell wall and cell
morphology - Other microscopy methods allow gathering of different information
or increase the resolution - May used different forms of staining, optical mechanisms, or both to
increase contrast - Include phase contrast, dark field, fluorescent, differential
interference, atomic force, confocal, transmission electron, and
scanning electron microscopy
18
Q
Phase-contrast
A
- Two parts of the light beam are integrated
- One comes straight through the specimen
- The other is highly diffracted light collected from the edge of the
lens - Light is diffracted due to the fact that various intra- and extracellular structures have different refractive indexes
- Diffracted light subtracts from direct light giving the structures
with the greatest refractive index the darkest appearance
19
Q
Dark-Field Microscopy
A
- An opaque disk over middle of light source creates a donut-shaped
beam. - Only way for light to reach the specimen is to approach at an angle -
normally miss lens - Only light reflected by the specimen itself enters the lens
- Specimen appears light against a dark background
