Lecture 8 part 2: Optics Flashcards Preview

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1

What limits brightfield microscopy?

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

2

What has enabled this to be surpassed?

Subresolution Optical Imaging using Localisation Microscopy

3

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

4

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

5

What is another reason for sub resolution imaging?

Lots of biological structures are near the resolution limit
- genes
- Macromolecule proteins RyR
- Proteins, individual channels

6

What is the abbe limit?

It is the diffraction limit of the microscope.

7

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

8

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)

9

What does localisation accuracy depend on?

Signal to noise ratio

- Statistical noise
- Background noise

10

How would you seperate flurophores using localisation?

Spectrally - No, limited number of channels


Time! use switchable dyes and stochastic switching

11

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)

12

Describe PALM;

Sample
- Activate
- Measure
- Bleach
- Repeat

= Paints result


Relies on switchable dyes

13

What are some switchable dyes?

Fluorescent proteins

Organic Dyes

There are many

14

What are some organic dyes?

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

15

How can FPs be programmed?

Photoactivation
Photoconversion
Photoswitching

16

What is photoactviation and example?

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

17

What is photoconversion and example?

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

18

What is photoswitching and examples?

Dark Bright
Reversible
DRONPA, rsFastLime, Padron

19

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

20

How do Rhodamineand Oxazine Derivatives function as indicators?

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

21

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

22

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.

23

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

24

Can we use localisation in live cell imaging?

Yes and we merry it with confocal to do so

25

What are some practical considerations for confocal microscopy?

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

26

What are some down sidez labelling tags?

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

27

Summery;

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