lecture 2 Flashcards
Anton von leeuwen hoek
- first person to see bacteria
- built many single lens microscopes
- achieved magnification of 200x
what did AVL call the living protozoa and bacteria he observed through his miscropcope
- animalcules
what was the structure of AVLs microscope
- finely polished glass lens between brass plates
modern compound micorscop
- has a light source
- has a condenser that focuses the light on the specimen that is on a slide
- there is magnification from the objective lens that collects light after it has passed through the specimen
- reaches the ocular lens that focus’s the light on the eye
what is the magnification of a typical compound micrscope
40 to 1000x
what is the max magnification of a compound microscope
- 1000x
refractive structures and the compound microscope
- higher refractive index = darker
- lower refractive index = just white light, it is just passing through
resolution
- ablity to distinguish between two very closely positions obejctes as separate entities
- minimum distance between two separate objects at which they are still discernible as two seperate
- we want a really small resolution
what can be seen with a light microscope
- plant and animal cells, bacteria
- NOT viruses, globular proteins, small molecules, or atoms
what is the max resolutiionof a light microscope
- usually cannot observe objects that are less than 0.2 micrometers apart, o. r 200 nm, this is the limit of resolution
resolution equation
d = 0.61(lambda)/ Nsinalpha
N
refractive index between the specimen and the objective lens
- you can add things like water or oil that will have higher refractive indexes
- sin alpha isa half angle of light entering the objcetive
- a specimen that is close tot he objective wilhave a wide half angle which is optimal
how to get a low resolution
- reduce the numerator by using a shorter wavelength of light
- increase the denominator by using oil or water or liquid with a higher refractive index or increase alpha (how long or round the lens is)
numerical aperture
- Nsinalpha (higher = better, that reduces the denominator which makes the overall resolution smaller)
contrasts in light microscopy
- when a part of the cell (i.e. the nucleus) refracts more light, the portion of light that passes through that part of the cell will be slowed down a bit (a quarter wavelength) and that causes a bit of interferenceop
optimal interference in light microscopy
- when the two waves are completely out of phase, tan d there is interference that causes the production of dim lighght
by how much do cellular constituents with high refractive properties slow the passage of a light beam
- a quarter wavelength
phase contrast microscopy
- still light microscopy
- used to examine live, unstained cells
- small differences of refractive indexes in cell and thicknesses are exploited and converted into contrast visible to th eye
process of phase contrast microscopy
- light from lamp
- annular diaphragm turns light into a cone of light, the big beam iss separated into a cone
- the cone is focused via a condenser lens
- part of cell with higher refractive properties will bend the light a little
- this causes the 1/4 wavelength slow down
- that light is passed through a phase plate
- the diffracted quarter slowed light is diffracted towards the centre part of the palte
- this slight is slowed down another quarter length
- ## refracted light is directed towards the center
what does the total 1/2 wavelength slowed do?
- gives more detail of intracellular components
issue with phase contrast microscopy
- for rounded cells, at periphery, refracted light can be accelerated instead of slowed
- ## that doubles the amplitude of the peaks
DIC microscopy
- examine live unstained cells
- small differences in refractive index and thickness within the cell ar converted into contrast that is visible to the eye.
- this uses polarized light, the microscope has polarizers
- more defined outline of large organelles such as nucleus and vacuole = better detail of cell edge
fluorescence microscopy
- uses fluorescence
fluoresce
emit visible light when absorb light at a specific wavelength
benefits of fluorescence microscopy
- visualize more than one protein or cell strucutre
fluorochromes
- usd in fluorescent micrsocopy
- absorb electrons to cause the outer oribital to be excited to a higher state
- when the electrons drop to its normal orbital, visible light released, fluorescenee
stokesshigt
- different between excitation and emission optima
- different fluorochromes have different optimal
= doesn’t use a light bulb, uses an intensee light source like a laser
fluorochrome colours
- wide vareity
- have different excitation and emission wavelentghs
- allows for labelling of more than one protein or organelle at the same time
examples of chemicals linked to flurocochromes that are able to stain cell structures nd organelles
DAPI
mitrotracker Red
Rhodmaine labelled phalloidin
DAPI
- stain nuclei blue
- high affinity to DNA
-fluroeces blue - binds well to nucleo
mitotracker red
- gets caught in michondiral inter membrane space
rhodamine labelled phalloidin
- stain mitochondria or actin filaments red
immunofluorescent staining
- conjugating a dye with antibodies to localize molecules of interest
fluorescent imaging in live cells
