Lecture 8 part 2: Optics Flashcards
What limits brightfield microscopy?
The diffraction limit which is a function of NA and PSF and consequently limits resolution.
What has enabled this to be surpassed?
Subresolution Optical Imaging using Localisation Microscopy
What is pivotal in the sub resolution imaging?
STED (Stimulated Emission Depletion)
This is a flurophore that forms a very tight PSF (20nm resolution)
This is essential in localisation microscopy?
Check this
Why bother with sub resolution imaging if EM already exists?
- Fluorescence has good contrast
- Colour or multichannel
- Flexibility & ease of labelling/preparation
- Living specimens
- Thick 3D specimens
What is another reason for sub resolution imaging?
Lots of biological structures are near the resolution limit
- genes
- Macromolecule proteins RyR
- Proteins, individual channels
What is the abbe limit?
It is the diffraction limit of the microscope.
What are some abbe equations?
FWHM(x) = 0.51 (wavelength) / NA
FWHM(z) = 1.67(n) (wavelength)/NA^2
Basically users PSF to determine resolution
This was laster refined by releigh
In lay terms how does localisation work?
Center of intensity reflects centre of PSF
If this isnt quite right fit a gussian
Allows for subresolution identification of PSF (i.e where two PSF couldnt be resolved, this localisation can seperate them)
What does localisation accuracy depend on?
Signal to noise ratio
- Statistical noise
- Background noise
How would you seperate flurophores using localisation?
Spectrally - No, limited number of channels
Time! use switchable dyes and stochastic switching
What is stochastic switching?
Switchingis on/off (a dye switches or it doesn’t)
Probabilityof switching can be made small (e.g. by lowering activation intensity)
Photoactviation switches dyes on therefore after many cycles can build an image using the localisation
(Photo activated localisation microscopy PALM)
Describe PALM;
Sample
- Activate
- Measure
- Bleach
- Repeat
= Paints result
Relies on switchable dyes
What are some switchable dyes?
Fluorescent proteins
Organic Dyes
There are many
What are some organic dyes?
Carbocyanineswitching e.g. Cy5
Rhodamineswitching e.g. Alexa 488
Engineered switches (caged/photochromiccompounds
How can FPs be programmed?
Photoactivation
Photoconversion
Photoswitching
What is photoactviation and example?
Dark -> Bright
Irreversible
PA-GFP, PA-CFP, PA-mCherry
What is photoconversion and example?
Eg. Green -> Red
Usually irreversible
mEos, Kaede
What is photoswitching and examples?
Dark Bright
Reversible
DRONPA, rsFastLime, Padron
How do carbocyanine dyes work?
E.g. Cy3-Cy5 pairs for STORM
E.g. Alexa 647 for direct STORM
Entry to dark state by covalent attachment of thiol, eg. mercapto-ethyl amine (MEA)
Exit by short wavelength light, thermal oxidation, or energy transfer from activator dye.
Switching improved by oxygen removal
How do Rhodamineand Oxazine Derivatives function as indicators?
Switching based on different oxidation states
Dark state partially reduced radicalEntry by reduction of triplet (usually via a ThiolegMEA)
Exit by thermal oxidation, or optically
Atto655
How can multiple colours be used?
Multi-colour localisation microscopy
Either sequential
- Measure red fluorophore, then green
- Issues with bleaching & crosstalk
Or simultaneous
- Use ratio to categorise fluorophores
Whats the hindrance with 3 d localisation?
Dye molecules switch & bleach so can’t take a normal z-stack
Need to get all information for 3D position at the same time.
Whats the solution for 3D localisation?
Record multiple images at different defocus
Change PSF so lateral shape gives information on defocus
3D position information from single frame
Number of methods:
- Astigmatism
- Double helix
- Phase ramp
Can we use localisation in live cell imaging?
Yes and we merry it with confocal to do so
