what is resolution, and what is it limited by?
resolution is the ability to distinguish two separate objects apart from one another; resoultion is limited by diffraction
Draw the PSF, airy disk, and where the beam of light and blurriness come from
How do you get super res?
Attempting to break the PSF (theoretical resolution/diffrraction imaging limit): The smallest spot the convex lens can focus cannot be less than the wavelength of light (PSF)
So how do you improve PSF?
Improving RESOLUTION
This depends on optical abberation, and the diffraction limit, the spatial resolving power of the optical lens (NA) and the wavelength of light you’re imaging
how do you improve super res image quality?
Increase NA angle on the objective lens. When maintaining the specimen, use glss or emersion oil as it has a bigger RI than air or water!
Explain how STED works, what does it stand for, what do you have to add to a confocal microscope to make it a STED microscope?
Stimulated electron depletion.
You add a second excitation beam (the stimulated excitation pulse) ns after the initial fluorescence excitation pulse. This bleaches the area around the fluorophore, meaning that you only scan (image) one fluorophore at a time rather than groups of fluorophores (because res is too low to distinguish between the two fluorophores). Thus even if your image is blurry, you know its only coming from one molecule, so you can deconvolute the image.
Outline the pros and cons, and the max resolution, of STED
pros
- based on confocal point scanning
- engineers excitation spot with illumination pattern
- takes seconds to take image
- reaches 3D res of up to 50nm
- can be used on live cells
cons
- limited to certain fluorophores
- photobleaching major concern
- requries careful calibration
- only images 2 colours
- expensive
How might you increase res of the widefield telescope, to super res? (100nm lateral, 60 after deconvolution)
Use SIM (sructured Illumination Microscopy)
- this projects illumination patterns onto the sample based on Moire pattern
- depends on 5 phase shifts in each direction; taking 3-5 images in each
- only one beam of excitation
- requires algorithmic image reconstruction
What are the pros and cons of SIM?
- based on widefield so cheaper
- less photobleaching than STED –> good for live samples!
- can image multiple colours
- can be used on live cells
weaknesses:
- STED better at 3D imaging, SIM is limited - better for 2D imaging
- grid illumination can cause artefacts
- local media environment dependent
- expensive
What do both PALM and STORM rely on?
Controlling the photoactivation of fluorophores –> gives rise to single molecule localisation
Outline the process of PALM
depends on photobleaching of fluorophors
you shine one low intesnity laser twice, imaging in between.
first time you shine the low power excitation laser the fluorophores are activated, so you image them and map the centroids (centre of each fluorophore), then you shine laser again and these fluorophores will bleach, so you image again and you can complete the process knowing the same fluorophores won’t be activated twice.
Idea is that you image long enough to bleach the fluorophres
Can be used with Alexa fluoro or GFP (endogenous fluorophores)
Outline the process of STORM, making comparisons to PALM
depends on single molecule localisation via photoblinking (vs photobleaching). e move through the energy levels with control of UV lasers
laser excites e from ground state to higher energy state. if laser activation is prolonged, e will transition to triplet to dark state. this is when the e blinks, and you image the sample to visualse which e are blinking. transition from triplet to dark state (ie blinking) is due to redox reactions.
strong 405 laser makes e go back to fround state, cycle can be recycled hundreds of time to put together images
depends on antibodu-labelled proteins
Why is a buffer so important in STORM?
- to prevent photobleaching
- also maintains pH for redox reaction (t state –> d state; UV light rescues e back to g state - inverse redox)
- thus buffer stabilises fluorophres and maintains blinking populations
what are the pros/cons of PALM/STORM?
pros:
- 20nm resolution
- cheaper than SIM/STED as based on widefield
- can image 3 colours
- PALM only - single molecule tracking
cons:
- cant use on live samples
- fixed and thin samples only
- aquisition time is long due to laser activation process
- STORM is largely dependent on the sample/buffer interaction
- only works on certain fluorophores
What method would you use to visualise proteins?
maybe CryoEM, or PALM/STORM (20nm res) but for this sample would have to be fixed and in a special buffer
What would you use to view a mammalian cell?
confocal or widefield . widefield has higher contrast so better for overall structure, and can be used for live samples
Would STED be suitable for visualising a synaptic vesicle?
Yes because you can view live samples, so you could watch synaptic transmission of labelled neurotransmitters. also res is 50nm.
But to see interactions between proteins that make up the axons of a neauron you might want to use PALM as res is 20nm
Give an exmaple of when you would use STED, SIM or PALM/STORM
STED –> visualising synaptic plasticity –> eg in dopamine pathways in addicted rats
Can also vsualise 3D images
PALM –> single molecule tracking eg proteins through a cascade, or visualise movement of electrons in electron transport chains
STORM –> synaptic protein interactions (nm level!!)
Outline the process of expansion microscopy
it involves expanding the sample in 3D with nanoscale resolution –> makes the sample physically bigger rather than using manificaqtion.
- (fixation) and label/stain sample with fluoro dye - this becomes anchored in the polymer matrix when the fluoro antibodies attach to the dye (anchoring)
- gelation and mechanical homogenization –> add diugestion buffer
- expansion with water
what are the pros and cons of expansion microscopy?
pros: expands the sample 3-10x published res 25nm applicable to both live tissue and clutured cells cheaper than super-res imaging
cons:
depends on labelling efficiency (antibodies binding to dyes)
fluorophores get degraded
gel handling –> careful not to deform structure
fluoresence reduces proportionally with volume expansion
sig increase of imaging and data load
what is the x-y resolution of widefield?
200nm
what is the x-y resolution of confocal?
180nm
what is the x-y resolution of SIM?
100nm
what is the x-y resolution of STED?
50nm
