Sup res microscopy Flashcards
(19 cards)
Describe super resolution microscopy
Em higher res than lm
Microscope resolution, near field microscopy
Trif
Visualizing single molecules
Palm/storm
Describe resolution
Limit of resolution - (d) = smallest distance between 2 points that can be distinguished
1/4th micron at most using light
Describe flureosnce image of flat cell
Microtubules = 25nm in diameter
Problem if these 2 close together = wont be Able to see detail
MTS = 250nm in diameter =look about 10x diameter bc blurred by diffraction light, very long and can see but if too
Describe near field microscopy = gen/technique
Betzig
Optical fibre with 30nm tip moved slowly over sample
Evaded diffraction limit and enabled super resolution fluorescence imaging
Describe near field microscopy = is it useful
Image acquitision slow = 30mins/image
Impractical for biological use
Okay for materials for science
Describe near field microscopy = image
Illuminate and move time over sample
30 mins
Resolution not limited by refraction
Works if sample flat
Describe what betzig did - nile red
Each dot in image = single molecule of fluroscnent - nile red
Can see bc far apart
Looked at in mathetical way = localize dot, turn on a few and turn off a few = build image = res limited by how well can localize molecules (50-40nm, determine centre, localize depending on amount of noise)
Describe image of molecules
Can visualize individual molecules
More difficult
Special camera
Dynein moving on mts
Better cam, (tirf to reduce background, very intense illumination)
Storm=
Stochastic optical reconstruction microscopy
What is storm = explain technical
Sample placed in special buffer (with reducing agent) that drives most of fluorescent due molecules into a dark state
Few remaining molecules distant from each other and can be individually visualized - intense illumination with very good cam (when molecules oxidized = visible)
Molecules randomly flicker into and out of dark state
Images taken about 10/sec for 15 mins = eventually visualize most o f molecules, localize them to 30nm
Image assembled showing molecule locations- using computer
Describe storm image
1 frame movie, over 15 mins
Molecules flick on and off
Then computer = makes up individual coated pits
Helps localize individual molecules
Palm stands for
Photo activation localization microscopy
What is palm similar to
Similar to storm - but works with special photoactivatable gfps = diff in how molecules vsiauzlied - turned on and off
Describe palm technique
Small proportion of paGFP = can be turned on by near ultraviolet light - uv - imaged and bleached= becomes visible if uv
Others can be turned on after = with prices repeated ~1000 times
First point localization technique
what is palm/storm not compatible with
Not compatible with confocal microscopy
Bc confocal not good at very dim images
what is palm/storm best with
Thin samples
So that faint signal from single fluroscent molecules is not swamped by autofluorescence - use of trif can help bit limits observation to bottom of samples
Can palm/storm be used in ‘em
Often used on sections prepared for ‘em= possible (embed photoactivatable gfp in palstic but very hard)
Can substitute for immunogold if willing to image same section on 2 microscopes
Can palm/storm be used in living cells
Very difficult but not impossible with living cells,
Does not work in sections
Other super-resolution techniques (less resolution than PALM/STORM, but easier to use)
Structured illumination - doubled resolution
Sted - works with confocal, high res, complicated
Airydisk - works with Confocal, easier
