Lecture 8 part 2: Optics

Question 1

What limits brightfield microscopy?

Answer 1

The diffraction limit which is a function of NA and PSF and consequently limits resolution.

Question 2

What has enabled this to be surpassed?

Answer 2

Subresolution Optical Imaging using Localisation Microscopy

Question 3

What is pivotal in the sub resolution imaging?

Answer 3

STED (Stimulated Emission Depletion)

This is a flurophore that forms a very tight PSF (20nm resolution)

This is essential in localisation microscopy?

Check this

Question 4

Why bother with sub resolution imaging if EM already exists?

Answer 4

• Fluorescence has good contrast
• Colour or multichannel
• Flexibility & ease of labelling/preparation
• Living specimens
• Thick 3D specimens

Question 5

What is another reason for sub resolution imaging?

Answer 5

Lots of biological structures are near the resolution limit

• genes
• Macromolecule proteins RyR
• Proteins, individual channels

Question 6

What is the abbe limit?

Answer 6

It is the diffraction limit of the microscope.

Question 7

What are some abbe equations?

Answer 7

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

Question 8

In lay terms how does localisation work?

Answer 8

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)

Question 9

What does localisation accuracy depend on?

Answer 9

Signal to noise ratio

• Statistical noise
• Background noise

Question 10

How would you seperate flurophores using localisation?

Answer 10

Spectrally - No, limited number of channels

Time! use switchable dyes and stochastic switching

Question 11

What is stochastic switching?

Answer 11

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)

Question 12

Describe PALM;

Answer 12

Sample

• Activate
• Measure
• Bleach
• Repeat

= Paints result

Relies on switchable dyes

Question 13

What are some switchable dyes?

Answer 13

Fluorescent proteins

Organic Dyes

There are many

Question 14

What are some organic dyes?

Answer 14

Carbocyanineswitching e.g. Cy5
Rhodamineswitching e.g. Alexa 488
Engineered switches (caged/photochromiccompounds

Question 15

How can FPs be programmed?

Answer 15

Photoactivation
Photoconversion
Photoswitching

Question 16

What is photoactviation and example?

Answer 16

Dark -> Bright
Irreversible
PA-GFP, PA-CFP, PA-mCherry

Question 17

What is photoconversion and example?

Answer 17

Eg. Green -> Red
Usually irreversible
mEos, Kaede

Question 18

What is photoswitching and examples?

Answer 18

Dark Bright
Reversible
DRONPA, rsFastLime, Padron

Question 19

How do carbocyanine dyes work?

Answer 19

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

Question 20

How do Rhodamineand Oxazine Derivatives function as indicators?

Answer 20

Switching based on different oxidation states
Dark state partially reduced radicalEntry by reduction of triplet (usually via a ThiolegMEA)
Exit by thermal oxidation, or optically
Atto655

Question 21

How can multiple colours be used?

Answer 21

Multi-colour localisation microscopy

Either sequential

• Measure red fluorophore, then green
• Issues with bleaching & crosstalk

Or simultaneous
- Use ratio to categorise fluorophores

Question 22

Whats the hindrance with 3 d localisation?

Answer 22

Dye molecules switch & bleach so can’t take a normal z-stack
Need to get all information for 3D position at the same time.

Question 23

Whats the solution for 3D localisation?

Answer 23

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

Question 24

Can we use localisation in live cell imaging?

Answer 24

Yes and we merry it with confocal to do so

Question 25

What are some practical considerations for confocal microscopy?

Answer 25

Switchable fluorophores
Strong laser ~300 mw for STORM
Sensitive detector (egEMCCD / sCMOSCamera)
High labelling density
Drift correction
Analysis software

Question 26

What are some down sidez labelling tags?

Answer 26

• Nanobodies-1/10 size
• SNAP-tag
• DNA paint

Question 27

Summery;

Answer 27

Diffraction no longer a limit to resolution
Localisationmicroscopy allows fundamental (practically ~10 fold) improvement in imaging resolution
- Uses switchable dyes
- And stochastic switching

Separate individual molecules in Time