Chapter 1 - Cells and Microscopy Flashcards
What is the maximum magnification of a Light Microscope?
x1500
What is the resolution limit of a Light Microscope?
200nm
What is resolution?
The ability to distinguish between 2 separate points.
The limit of resolution = λ / 2
What is the resolution limit of a CLSM?
200nm
Advantages of Light Microscopes
- Low skill set needed
- Cheaper than other types
- Can be transported to use in field work
- Can observe living organisms
Disadvantages of Light Microscopes
- Low resolution
- Limited magnification
- Many internal structures can’t be seen
How to prepare a temporary slide for observation using Light Microscope.
- Fixation - use 70% alcohol
- Staining - use a few drops of differential stain
- Mounting - cover with coverslip to exclude dust and air.
Advantages of temporary slides procedure
- Rapid and Simple
- Can mount in glycerine to prolong examination period.
How to prepare a permanent slide for observation using Light Microscope
- Fixation - preserves specimen, minimises distortion, alcohol or acestic acid added.
- Dehydration - place in up to 70% alcohol.
- Clearing - addition of xylol
- Embedding - resin, plastic or wax
- Sectioning - use a microtome
- Differential staining - can be permanent or temporary.
- Mounting - embeds and protects material.
Different types of plant stain
Iodine - temporary, blue-black, starch
Aniline Sulfate - temporary, yellow, lignin
Eosin - permanent, red, cellulose
Toludine -permanent.
Red, lignin.
Purple, cellulose
Advantages of using differential stains
- easier to observe tissues, cells and chemicals
- allows to distinguish between different vessels in plants.
- allows to distinguish between different types of white blood cells.
How to use Leishman’s stain
- Allow blood smear to airdry
- fix with methanol
- flood with leishman’s stain
- leave for 2 mins
- dilute with distilled water
- leave for 7-10 mins
- wash with distilled water until appears pale pink.
How to use Wright’s stain
- Allow blood smear to air dry
- flood with wright’s stain
- leave for 3-4 mins
- dilute with distilled water
- leave for 6-8 mins
- wash with distilled water or phosphate buffer until has pale pink edges
- air dry and mount
key points of cell theory
- a cell is the basic unit of all life
- all living organisms are made up of either one or more cells
- metabolic processes occur in living cells
- all new cells come from pre-existing cells
- cells possess genetic material which is passed to daughter cells.
- a cell is the smallest unit of life capable of surviving independently.
Advantages of electron microscopes
- greater resolution
- higher magnifications can be used
- finer detail can be seen
Disadvantages of electron microscopes
- specimen must be dead
- highly expensive
- high skill set required
- must be used in specialized room
- more complex process
- more prone to artifacts
Advantages of using a CLSM
- ability to eliminate or reduce background information
- high resolution
- can obtain 3D reconstructions
- can image living cells and tissues
- any out of focus light is blocked
How are fluorescent markers work?
- fluorochromes attach to antibody that is specific for one antigen on or in the cell
- can also be tagged to a chemical
- each fluorochrome has its own peak excitation and emission wavelength
- lasers with different wavelengths are used for different fluorochromes
- laser excites the fluorochrome causing the tagged cells to fluoresce
- fluorescence is counted by the detector
What is the maximum resolution of an electron microscopes?
0.1nm
What is the maximum resolution of a CLSM?
x2000
How is the beam focused in a light microscope?
Condenser
How is the beam focused in a CLSM?
Objective lens
How is the beam focused in electron microscopes?
Electromagnets
Maximum magnification of TEM?
x1,000,000
What is the maximum magnification of an SEM?
x500,000
