6 - Advances in Light Microscopy - Hwang Flashcards
What are the units used to measure bacteria and similar structures etc. Give all of the prefixes
nm
um
mm
m
give rough estimates of the sizes of E. coli and a bacteriophage
E. coli; 1-2um diameter (microns)
Bacteriophage; 200nm
what type of microscopes are used to visualise atoms?
electron microscopes
draw a diagram showing the anatomy of a light microscope. explain this briefly
- light source shined on the sample, this is directed onto the sample by the condensory
- light is scattered by the sample and collected by the objective lens
name the 3 important factors of light microscopy and explain them briefly
MAGNIFICATION; combination of microscope objectives and ocular lens (eyepiece). 10,20,40,60,100x
RESOLUTION; numerical aperture (NA) of the objectives. for light microscopy, anything smaller than bacteria wil not be visualised
CONTRAST; ability to distinguish between sample and background. Phase contrast (light microscopy) and fluorescence (light microscopy)
what is the actual number for the resolution limit for a light microscope
- half the wavelength of light
- around 0.2um
what is Abbe’s law? give the eqn
gives the resolution limit of a microscope
d = lamda / 2NA
where d = aperture angle
what is the numerical aperture?
resolving power of the objectives
for a fixed resolution, what occurs when 2 points come closer together?
then they appear as one if the resolving power does not change
what are the advantages of using fluorescence microscopy?
- high contrast to background when the structures absorb and emit light
- multicolour imaging by labelling different molecules eg proteins, DNA
- allows spatial and temporal distribution tracking
- non invasive
state what is shown in q11 of 328 - 6 word
- what do the different colours represent?
shows metaphase of mitosis
green = MTs
blue = condensed chromosomes
pink = kinetochore
describe the process of a molecule showing fluorescence
- energy from light source eg UV, laser causes excitation of electrons within the fluorophore to a higher energy level
- relaxation back to ground state through various vibrational energy levels emits light
draw a simple Jablonski diagram showing a molecule being excited and then returning to the ground state
328 - 6 word
what is Stokes’ shift? draw a digram of this
when we have absorption of a shorter wavelength which then shifts to emit light at a longer wavelength
what is the relationship between energy and wavelenght? why does this play a role in fluorescence?
- amount of energy is inversely proportional to wavelength therefore the shorter the wavelength the larger the energy of the photon
- this is seen when we have absorption of a shorter wavelength giving rise to a larger energy. then the loss of this energy during emission
give 3 examples of fluorophores
fluorescin, Rhodamine B, Alexa 647
give the overall strucuture of flurophores
- many aromatic rings with a delocalised e- system. hence why we can get excited states
what happens when we excite a larger flurophore compared to a smaller one
more excitation, larger stoke shift therefore more colours seen on the return to ground state
what fluorophore can be used for tagging proteins? give the origins of this
- GFP used to tag proteins through fusing with another gene. the protein will then fluoresce if expressed
- firstly isolated from a jellyfish
what is the name of the technique used to break the resolution limit? give the resolution this technique allows us to see to and give an example
SUPER RESOLUTION LIGHT MICROSCOPY
- allows 30nm resolution
- can see individual peptidoglycan structures on S. aureus
name the 2 super resolution techniques and describe them
PALM; PhotoActivationLocalisationMicroscopy
STORM; STochasticOpticalReconstructionMicroscopy
- both use stochastic (random) localisations
- both turn on stochastically sparse subsets of flurophores, separating their emission time so we can get individual frames which can be mapped on to each other giving the overall structure
- PALM uses photactivatable fluorescent proteins and continuous cycles of photoactivation and photobleaching
- STORM uses organic dyes eg Alexa 647 and Cy5 that reversibly switch on and off
- density of activated molecules kept low therefore no overlap
in 328 - 6, identify the proteins that are fluorsecently tagged and give the names of the species that they are present in
a) MreB filaments in B. subtilis
b) FtsZ ring in B. subtilis
